Compositions and methods for treating and diagnosing irritable bowel syndrome

ABSTRACT

Compositions and methods for diagnosing and treating CVH and CVH-associated disorders are disclosed. Genes differentially expressed in CVH tissues relative to normal tissues are identified. The genes and the gene products (i.e., the polynucleotides transcribed from and polypeptides encoded by the genes) can be used as markers of CVH. The genes and the gene products can also be used to screen agents that modulate the gene expression or the activities of the gene products.

This application claims priority from U.S. Provisional Application Ser.No. 60/496,716 filed Aug. 21, 2003. The entirety of that provisionalapplication is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to the diagnosis and treatmentof disorders associated with chromic visceral hypersensitivity (CVH),and in particular irritable bowel syndrome (IBS). The invention alsorelates to genes associated with CVH, polynucleotides transcribed fromthese genes and polypeptides encoded by these genes. Suchpolynucleotides and polypeptides can be used for the diagnosis andtreatment of CVH.

BACKGROUND OF THE INVENTION

Irritable Bowel Syndrome (IBS) is a functional bowel disorder of unknownetiology. A functional disorder refers to a disorder or disease wherethe primary abnormality is an altered physiological function, ratherthan an identifiable structural or biochemical cause. IBS ischaracterized by a group of symptoms including intermittent abdominalpain and discomfort and alterations in bowel habits, such as loose ormore frequent bowel movements, diarrhea, and/or constipation that occurin the absence of detectable ongoing organic disease.

IBS affects approximately 10-20% of the general population. It is themost common disease diagnosed by gastroenterologists and one of the mostcommon disorders seen by primary care physicians. IBS is understood as amulti-faceted disorder. In people with IBS, symptoms result from whatappears to be a disturbance in the interaction between the gut orintestines, the brain, and the autonomic nervous system that altersregulation of bowel motility (motor function) or sensory function.

Human studies demonstrate that IBS is associated with a state of chronicvisceral hypersensitivity (CVH) suggesting that processing of visceralsensory information is altered. However, little is known about how theafferent nervous system is changed in this syndrome. A hallmark of TBSis increased visceral hypersensitivity, but the molecular changesunderlying the development and maintenance of chronic visceralhypersensitivity in IBS are not known. Current medical treatments forIBS primarily target peripheral symptoms rather than the underlyingcauses, and therapeutic gains from drug treatments are usually modestand the placebo responses are high (Mertz et al., Gastroenterology,109:40-52, 1995). Defining the underlying neurological and moleculardefects is therefore important to the design of more successfultherapeutic strategies. Moreover, there is a need in the art forimproved methods for screening, diagnosing, and treating MS and otherCVH-related disorders.

CNI-1493 is a MAPK and TNF inhibitor with anti-inflammatory and possibleanalgesic actions. CNI-1493 inhibits signal transduction pathways bypreventing phosphorylation of p38 MAP kinase and JNK, and inhibitsproduction of the proinflammatory cytokines such as TNF-alpha, IL-1,MIP-1 alpha, and MIP-1 beta. In animal models, CNI-1493 has shownprotective activity against a wide variety of conditions, ranging fromstroke to inflammatory bowel disease. However, CNI-1493 has never beentested for its anti-nociceptive activity in the absence of inflammation.Recently, an animal model of chronic visceral hypersensitivity wascreated using mechanical and chemical irritation of the colon ofneonatal rats (Al-Chaer et al., Gastroenterology, 119:1276-1285, 2000).The animal model provides an ideal platform for studying IBS, validatingthe neurogenic components of functional abdominal pain, and testingagents that may reduce visceral hypersensitivity.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to the treatment fordisorders associated with CVH using guanylhydrazone. In one embodiment,the present invention provides a treatment for IBS using CNI1493.

Another aspect of the present invention relates to CVH-related genes(CVHGs) and the gene products, which include the polynucleotidestranscribed from the CVHGs (CHVPNs) and the polypeptides encoded by theCVHGs (CHVPPs).

In one embodiment, the present invention provides methods for diagnosingand monitoring CVH and CVH-related disorders by comparing the expressionlevels of one or more CVHGs at the nucleotide or protein level inbiological samples from a subject to control samples.

In another embodiment, the present invention provides pharmaceuticalcompositions for the treatment of CVH and CVH-related disorders. Thepharmaceutical compositions comprise a pharmaceutically acceptablecarrier and at least one of the following: (1) a CVHG product; (2) anagent that modulates an activity of a CVHG product; and (3) an agentthat modulates the expression of a CVHG.

In another embodiment, the present invention provides methods fortreating CVH and CVH-related disorders in a patient with thepharmaceutical compositions described above. The patient may beafflicted with CVH, in which case the methods provide treatment for thedisease. The patient may also be considered at risk for CVH, in whichcase the methods provide prevention for disease development.

In another embodiment, the present invention provides methods forscreening anti-CVH agents based on the agents' interaction with CVHPPs,or the agents' effect on the activity or expression of CVHPPs.

In another embodiment, the present invention provides biochips fordiagnosing CVH and CHH-related disorders, and for screening agents thatinhibit CVH. The biochips comprise at least one of the following (1) aCVHPP or its variant, (2) a portion of a CVHPP or its variant (3) aCVHPN or its variant, and (4) a portion of a CVHPN or its variant.

In another embodiment, the present invention provides a kit fordiagnosing CVH and CVH-related disorders. The kit comprises at least oneof the following (1) polynucleotide probe that specifically hybridizesto a CVHPN, and (2) an antibody capable of specific binding to a CVHPP.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Increased sensitivity to CRD in adult rats treated with aceticacid on P10 (n=8). Data was analyzed by two-way repeated measures ANOVAwith distention pressure as the repeated factor and P10 treatment as abetween group factor. There was a significant effect of P10 treatment (F1, 12.98) p<0.003, of distention pressure (F 7, 89.9) p<0.001, and therewas a significant interaction between distention pressure and P10treatment (F 7, 4.04) p<0.001. Means were compared with a Tukey test.Significant differences between acetic acid treated and controls werefound at distention pressures of 30 (p=0.004), 40 (p<0.001), 50(p<0.001), 60 (p=0.001) and 70 (p=0.035) mm Hg.

FIG. 2. Effect of CNI-1493 on the response of sensitized rats to gradedCRD (n=8). Data was analyzed by two-way repeated measures ANOVA withdistention pressure as the repeated factor and drug treatment as abetween group factor. There was a significant effect of CNI1493treatment (F 1, 16.96) p=0.001, of distention pressure (F 7, 28.55)p<0.001, but there was no significant interaction between distentionpressure and CNI1493 treatment (F 7, 1.94) p=0.071. Means were comparedwith a Tukey test. Significant differences between CNI-1493 treated andcontrols were found at distention pressures of 20 (P=0.001), 30(p=0.003), 40 (p<0.001), 50 (p=0.001), 60 (p<0.001) and 70 (p=0.015) and80 (p=0.007) mm Hg.

FIG. 3. Colon histology and MPO activity. H&E stained colon sectionsfrom control, vehicle (A); control, cni-1493 (B); sensitized, vehicle(C); and sensitized, CNI1493 (D). Histogram showing MPO activity incolons (E).

DETAILED DESCRIPTION OF THE INVENTION

The preferred embodiments of the invention are described below. Unlessspecifically noted, it is intended that the words and phrases in thespecification and claims be given the ordinary and accustomed meaning tothose of ordinary skill in the applicable art or arts. If any othermeaning is intended, the specification will specifically state that aspecial meaning is being applied to a word or phrase.

It is further intended that the inventions not be limited only to thespecific structure, material or acts that are described in the preferredembodiments, but in addition, include any and all structures, materialsor acts that perform the claimed function, along with any and all knownor later-developed equivalent structures, materials or acts forperforming the claimed function.

Further examples exist throughout the disclosure, and it is notapplicant's intention to exclude from the scope of his invention the useof structures, materials, methods, or acts that are not expresslyidentified in the specification, but nonetheless are capable ofperforming a claimed function.

The present invention is generally directed to compositions and methodsfor the diagnosis, treatment, and prevention of CVH and CVH-relateddisorders; and to the identification of novel therapeutic agents for CVHand CVH-related disorders. The present invention is based on the findingthat guanylhydrazone is capable of ameliorating CVH in a rat model ofIBS and the discovery of transcribed polynucleotides that aredifferentially expressed in the colon tissue of rats with chemicallyinduced CVH relative to control animals.

DEFINITIONS AND TERMS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “a differentially expressed gene” refer to agene that meets all of the following criteria during an Affymetrixmicroarray analysis: (1) the average expression of the gene shows a foldchange of two or greater compared with controls and (2) significantchanges in gene expression were detected by analyzing signal intensityvalues by two-way ANOVA with 99% confidence. The differentiallyexpressed genes identified in the colon tissue samples of CVH andCNI1493-treated rats are designated as CVH-related genes (CVHGs). CVHGsgenerally refer to the genes listed in Tables 3-8.

As used herein, the terms “CVH-related polynucleotide (CVHPN)” and “CVHGpolynucleotide” are used interchangeably. The terms include atranscribed polynucleotide (e.g., DNA, cDNA or mRNA) that comprises oneof the CVHG sequences or a portion thereof.

As used herein, the terms “CVH-related polypeptide (CVHPP)” and “CVHGprotein” are used interchangeably. The terms include polypeptidesencoded by an CVHG, an CVHPN, or a portion of an CVHG or CVHPN.

As used herein, a “CVHG product” includes a nucleic acid sequence and anamino acid sequence (e.g., a polynucleotide or polypeptide) generatedwhen an CVHG is transcribed and/or translated. Specifically, CVHGproducts include CVHPNs and CVHPPs.

As used herein, a “variant of a polynucleotide” includes apolynucleotide that differs from the original polynucleotide by one ormore substitutions, additions, deletions and/or insertions such that theactivity of the encoded polypeptide is not substantially changed (e.g.,the activity may be diminished or enhanced, by less than 50%, andpreferably less than 20%) relative to the polypeptide encoded by theoriginal polynucleotide.

A variant of a polynucleotide also includes polynucleotides that arecapable of hybridizing under reduced stringency conditions, morepreferably stringent conditions, and most preferably highly stringentconditions to the original polynucleotide (or a complementary sequence).Examples of conditions of different stringency are listed in Table 2.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.

As used herein, a “variant of a polypeptide” is a polypeptide thatdiffers from a native polypeptide in one or more substitutions,deletions, additions and/or insertions, such that the bioactivity orimmunogenicity of the native polypeptide is not substantiallydiminished. In other words, the bioactivity of a variant polypeptide orthe ability of a variant polypeptide to react with antigen-specificantisera may be enhanced or diminished by less than 50%, and preferablyless than 20%, relative to the native polypeptide. Variant polypeptidesinclude those in which one or more portions, such as an N-terminalleader sequence or transmembrane domain, have been removed. Otherpreferred variants include variants in which a small portion (e.g., 1-30amino acids, preferably 5-15 amino acids) has been removed from the N-and/or C-terminal of the mature protein.

Modifications and changes can be made in the structure of a polypeptideof the present invention and still obtain a molecule having biologicalactivity and/or immunogenic properties. Because it is the interactivecapacity and nature of a polypeptide that defines that polypeptide'sbiological activity, certain amino acid sequence substitutions can bemade in a polypeptide sequence (or, of course, its underlying DNA codingsequence) and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is believed that the relative hydropathiccharacter of the amino acid residue determines the secondary andtertiary structure of the resultant polypeptide, which in turn definesthe interaction of the polypeptide with other molecules, such asenzymes, substrates, receptors, antibodies, antigens, and the like. Itis known in the art that an amino acid can be substituted by anotheramino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionof amino acids whose hydropathic indices are within +/−2 is preferred,those that are within +/−1 are particularly preferred, and those within+/−0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or polypeptide fragment, is intended for use inimmunological embodiments. U.S. Pat. No. 4,554,101, incorporatedhereinafter by reference, states that the greatest local averagehydrophilicity of a polypeptide, as governed by the hydrophilicity ofits adjacent amino acids, correlates with its immunogenicity andantigenicity, i.e. with a biological property of the polypeptide.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1);threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent polypeptide. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine (See Table 1, below). The present invention thuscontemplates functional or biological equivalents of an CVHPP as setforth above.

TABLE 1 Amino Acid Substitutions Exemplary Residue Original ResidueSubstitution Ala Gly; Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln AsnGlu Asp Gly Ala His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg Met Leu;Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

A variant may also, or alternatively, contain nonconservative changes.In a preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure, tertiary structure, andhydropathic nature of the polypeptide.

Polypeptide variants preferably exhibit at least about 70%, morepreferably at least about 90% and most preferably at least about 95%sequence homology to the original polypeptide.

A polypeptide variant also includes a polypeptide that is modified fromthe original polypeptide by either natural processes, such aspost-translational processing, or by chemical modification techniqueswhich are well known in the art. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. It will be appreciated that the sametype of modification may be present in the same or varying degrees atseveral sites in a given polypeptide. Also, a given polypeptide maycontain many types of modifications. Polypeptides may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic polypeptidesmay result from post-translation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a fluorophore or a chromophore, covalent attachment of aheme moiety, covalent attachment of a nucleotide or nucleotidederivative, covalent attachment of a lipid or lipid derivative, covalentattachment of phosphotidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.

As used herein, a “biologically active portion” of a CVHPP includes afragment of a CVHPP comprising amino acid sequences sufficientlyhomologous to or derived from the amino acid sequence of the CVHPP,which includes fewer amino acids than the full length CVHPP, andexhibits at least one activity of the CVHPP. Typically, a biologicallyactive portion of a CVHPP comprises a domain or motif with at least oneactivity of the CVHPP. A biologically active portion of a CVHPP can be apolypeptide which is, for example, 10, 25, 50, 100, 200 or more aminoacids in length. Biologically active portions of a CVHPP can be used astargets for developing agents which modulate a CVHPP-mediated activity.

As used herein, an “immunogenic portion,” an “antigen,” an “immunogen,”or an “epitope” of a CVHPP includes a fragment of a CVHPP comprising anamino acid sequence sufficiently homologous to, or derived from, theamino acid sequence of the CVHPP, which includes fewer amino acids thanthe full length CVHPP and can be used to induce an anti-CVHPP humoraland/or cellular immune response.

As used herein, the term “modulation” includes, in its variousgrammatical forms (e.g., “modulated”, “modulation”, “modulating”, etc.),up-regulation, induction, stimulation, potentiation, and/or relief ofinhibition, as well as inhibition and/or down-regulation or suppression.

As used herein, the term “control sequences” or “regulatory sequences”refers to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism. The term“control/regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Control/regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences).

A nucleic acid sequence is “operably linked” to another nucleic acidsequence when the former is placed into a functional relationship withthe latter. For example, a DNA for a presequence or secretory leaderpeptide is operably linked to DNA for a polypeptide if it is expressedas a preprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers are used in accordance with conventional practice.

As used herein, the “stringency” of a hybridization reaction refers tothe difficulty with which any two nucleic acid molecules will hybridizeto one another. The present invention also includes polynucleotidescapable of hybridizing under reduced stringency conditions, morepreferably stringent conditions, and most preferably highly stringentconditions, to polynucleotides described herein. Examples of stringencyconditions are shown in Table 2 below: highly stringent conditions arethose that are at least as stringent as, for example, conditions A-F;stringent conditions are at least as stringent as, for example,conditions G-L; and reduced stringency conditions are at least asstringent as, for example, conditions M-R.

TABLE 2 Stringency Condition Poly- Stringency nucleotide HybridHybridization Wash Temp. Condition Hybrid Length (bp)¹ Temperature andBuffer^(H) and Buffer^(H) A DNA:DNA >50 65° C.; 1xSSC -or- 65° C.; 42°C.; 1xSSC, 50% formamide 0.3xSSC B DNA:DNA <50 T_(B)*; 1xSSC T_(B)*;1xSSC C DNA:RNA >50 67° C.; 1xSSC -or- 67° C.; 45° C.; 1xSSC, 50%formamide 0.3xSSC D DNA:RNA <50 T_(D)*; 1xSSC T_(D)*; 1xSSC ERNA:RNA >50 70° C.; 1xSSC -or- 70° C.; 50° C.; 1xSSC, 50% formamide0.3xSSC F RNA:RNA <50 T_(F)*; 1xSSC T_(F)*; 1xSSC G DNA:DNA >50 65° C.;4xSSC -or- 65° C.; 42° C.; 4xSSC, 50% formamide 1xSSC H DNA:DNA <50T_(H)*; 4xSSC TH*; 4xSSC I DNA:RNA >50 67° C.; 4xSSC -or- 67° C.; 45°C.; 4xSSC, 50% formamide 1xSSC J DNA:RNA <50 T_(J)*; 4xSSC T_(J)*; 4xSSCK RNA:RNA >50 70° C.; 4xSSC -or- 67° C.; 50° C.; 4xSSC, 50% formamide1xSSC L RNA:RNA <50 T_(L)*; 2xSSC T_(L)*; 2xSSC M DNA:DNA >50 50° C.;4xSSC -or- 50° C.; 40° C.; 6xSSC, 50% formamide 2xSSC N DNA:DNA <50T_(N)*; 6xSSC T_(N)*; 6xSSC O DNA:RNA >50 55° C.; 4xSSC -or- 55° C.; 42°C.; 6xSSC, 50% formamide 2xSSC P DNA:RNA <50 T_(P)*; 6xSSC T_(P)*; 6xSSCQ RNA:RNA >50 60° C.; 4xSSC -or- 60° C.; 45° C.; 6xSSC, 50% formamide2xSSC R RNA:RNA <50 T_(R)*; 4xSSC T_(R)*; 4xSSC ¹The hybrid length isthat anticipated for the hybridized region(s) of the hybridizingpolynucleotides. When hybridizing a polynucleotide to a targetpolynucleotide of unknown sequence, the hybrid length is assumed to bethat of the hybridizing polynucleotide. When polynucleotides of knownsequence are hybridized, the hybrid length can be determined by aligningthe sequences of the polynucleotides and identifying the region orregions of optimal sequence complementarity. ^(H)SSPE (1xSSPE is 0.15MNaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substituted forSSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridizationand wash buffers; washes are performed for 15 minutes afterhybridization is complete. T_(B)* − T_(R)*: The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (T_(m)) ofthe hybrid, where T_(m) is determined according to the followingequations. For hybrids less than 18 base pairs in length, T_(m)(° C.) =2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.) = 81.5 ⁺ 16.6(log₁₀Na⁺) ⁺ 0.41(% G⁺C) −(600/N), where N is the number of bases in the hybrid, and Na⁺ is theconcentration of sodium ions in the hybridization buffer (Na⁺ for 1xSSC= 0.165M).

As used herein, the terms “immunospecific binding” and “specificallybind to” refer to antibodies that bind to an antigen with a bindingaffinity or 10⁵ Moles taken either from pre-CVH or from a subject whohas not suffered CVH, or from a cell, tissue or sample that is notaffected by CVH. Control samples of the present invention are taken fromnormal samples.

As used herein, the term “expression pattern” includes expression of agroup of genes at RNA or protein level, the quantity or activity of eachmember of which is correlated with the incidence or risk of incidence ofCVH and CVH associated diseases. An expression pattern comprisesexpression level of 2 or more CVHGs. An expression pattern may alsocomprise expression level of 2-5, 5-15, 15-35, 35-50, or more than 50CVHGs.

As used herein, the terms “treating,” “treatment,” and “therapy” referto curative therapy, prophylactic therapy, and preventative therapy.

Various aspects of the invention are described in further detail in thefollowing subsections. The subsections below describe in more detail thepresent invention. The use of subsections is not meant to limit theinvention; subsections may apply to any aspect of the invention.

One aspect of the present invention relates to a method for treating CVHand CVH-related disorders with compositions comprising a guanylhydrazonecompound. In one embodiment, the CVH-related disorder is IBS and theguanylhydrazone compound is CNI1493.

Another aspect of the present invention relates to CVH-related genes.Briefly, new born rats were sensitized by infusion of acetic acid intothe colon at P10. Control rats received saline. At eight weeks, theanimals were divided into four groups: control+vehicle; control+CNI1493;sensitized+vehicle; and sensitized+CNI1493. Colon samples were obtainedafter CNI1493 treatment (5 mg/kg for four days, the vehicle groupreceived vehicle only). Gene expression patterns in the colon tissuesamples and S1 dorsal root ganglia (S1 DRG) were analyzed usingAffymetrix rat genome 230A chips (Affymetrix, Santa Clara, Calif.).Single array analysis for each chip was performed by AffymetrixMicroarray Suite (MAS) software to produce a detection call, present,absent or marginal and a signal intensity value for each gene that is arelative measure of abundance of the transcript. For comparison ofsignal intensity values between chips, all chips will be scaled to anaverage intensity of 500. Genes called “Absent” across all chips andgenes without a Fold-change ≧2.0 in at least one of the pairwisecomparisons of chips from different treatment groups are excluded. Theprobe sets with absolute call “Absent” across all chips and Fold-change<2.0 in all of the possible pairwise comparisons, are filtered out.ANOVA was then performed on the filtered data set. Significant changesin gene expression were detected by analyzing signal intensity values bytwo-way ANOVA with 99% confidence limits. The differentially regulatedgenes were subjected to cluster analysis to identify genes associatedwith sensitization and treatment.

Tables 3 and 4 provides a list of the genes that are differentiallyexpressed in the colon and S1 DRG, respectively, of the sensitizedanimals, i.e., animals suffering from CVH. Tables 5 and 6 provide a listof the genes that are differentially expressed in the colon and S1 DRG,respectively, in CNI1493-treated animals. Since CNI1493 treatmentameliorates CVH in the sensitized animals, genes differentiallyregulated by CNI1493 may also be related to the etiology of CVH.Accordingly, genes listed in Tables 3-6 are designated as CVH-relatedgenes (CVHGs).

TABLE 3 Genes differentially expressed in sensitized colon No. Acce. No.Symbol CTRL-VHL CTRL-CNI IBS-VHL IBS-CNI 73 NM_021769 Sult-n 1.00 0.852.98 1.85 57 NM_022531 Des 1.00 1.03 2.55 1.31 14 BF389682 zzN/A 1.001.53 2.49 2.04 16 BF419200 Cebpd 1.00 1.03 2.44 2.28 26 BI296437 zzN/A1.00 1.14 2.43 1.21 25 NM_013002 Pcp4 1.00 0.88 2.29 1.14 55 NM_013122Igfbp2 1.00 0.75 2.19 0.94 74 NM_024400 Adamts1 1.00 0.90 2.11 1.14 65AI229029 Tubb3 1.00 0.93 2.05 1.53 43 BI282702 Acta2 1.00 0.95 2.03 0.9944 BF399310 zzN/A 1.00 0.64 2.01 0.78 64 NM_031970 Hspb1 1.00 1.14 1.901.40 12 ILGFBP4 BC019836 1.00 1.41 1.88 2.00 58 BF290193 zzN/A 1.00 0.651.84 0.90 36 AA012755 1.00 0.72 1.83 0.79 45 AI103600 zzN/A 1.00 0.571.81 0.58 63 AA851939 Fxyd6 1.00 0.95 1.81 1.26 56 AA799832 zzN/A 1.000.78 1.80 0.95 50 NM_019904 Lgals1 1.00 0.74 1.79 0.96 37 NM_017148Csrp1 1.00 0.63 1.72 0.71 35 BI283060 zzN/A 1.00 0.63 1.71 0.69 39AA800892 zzN/A 1.00 0.57 1.71 0.68 29 BG373779 LOC308709 1.00 0.48 1.680.59 2 BI285494 Ifitm31 1.00 3.86 1.66 4.90 47 NM_053770 Argbp2 1.000.79 1.66 0.77 34 NM_012893 Actg2 1.00 0.48 1.66 0.56 46 NM_130403Ppp1r14a 1.00 0.91 1.65 0.99 15 BG663107 zzN/A 1.00 1.06 1.65 1.23 30D29960 Loc192245 1.00 0.51 1.64 0.54 48 BM386598 zzN/A 1.00 0.90 1.600.95 71 AA818342 zzN/A 1.00 0.88 1.58 1.25 5 AW523747 Amigo2 1.00 0.881.57 2.10 40 AA817802 zzN/A 1.00 0.55 1.57 0.69 75 BE112887 zzN/A 1.001.06 1.55 1.05 33 M23764 Tpm1 1.00 0.60 1.54 0.68 59 AW522471 Exo70 1.000.65 1.54 1.00 8 EST 1.00 0.93 1.52 2.14 31 AI044427 zzN/A 1.00 0.591.52 0.69 41 BI279044 zzN/A 1.00 0.49 1.51 0.52 9 EST 1.00 1.48 1.512.10 28 BI291848 zzN/A 1.00 0.62 1.51 0.81 24 AI411809 zzN/A 1.00 0.891.51 1.06 67 NM_053440 Stmn2 1.00 0.79 1.49 1.06 54 BI285456 zzN/A 1.000.83 1.49 1.06 32 NM_031549 Tagln 1.00 0.54 1.47 0.64 38 AI177055 zzN/A1.00 0.79 1.47 0.82 52 BI279661 Wfdc1 1.00 0.66 1.46 0.94 20 AI229240zzN/A 1.00 0.68 1.46 1.69 1 BI285346 Ppp4r1 1.00 1.34 1.46 1.51 53NM_134449 Prkcdbp 1.00 0.87 1.45 1.01 49 BF555956 zzN/A 1.00 0.85 1.450.91 42 X16262 Myh11 1.00 0.54 1.44 0.60 51 BG666999 Slc25a4 1.00 0.791.44 0.94 70 AI177366 Itgb1 1.00 1.00 1.42 1.24 61 AA686007 Parva 1.000.80 1.41 0.99 68 BM389644 zzN/A 1.00 0.88 1.41 1.07 18 BG378721 zzN/A1.00 0.95 1.40 1.21 60 AA893484 Fn1 1.00 0.51 1.40 0.88 27 NM_019304Dgkb 1.00 0.67 1.31 0.86 7 EST 1.00 1.09 1.31 1.51 19 NM_017131 Casq21.00 0.71 1.30 1.19 6 U06434 Scya4 1.00 1.18 1.30 1.76 10 AB043636 Kcnj81.00 1.22 1.29 1.65 11 EST 1.00 0.82 1.29 1.55 66 AF081582 Evt1 1.000.85 1.29 1.03 3 NM_024145 Fgr 1.00 1.03 1.26 1.70 72 BI296312 zzN/A1.00 0.80 1.26 1.04 13 AA859496 Gch 1.00 0.93 1.24 1.34 22 BF394235zzN/A 1.00 0.55 1.23 0.99 69 BF551377 zzN/A 1.00 0.81 1.21 0.99 62AI179984 Dmrs91 1.00 0.49 1.21 0.70 21 NM_133605 Camk2g 1.00 0.77 1.181.01 17 U27518 LOC286989 1.00 0.50 1.11 1.19 4 NM_022688 Porf1 1.00 0.801.08 1.42 23 AW254190 1.00 0.66 1.05 1.03 87 BM386844 zzN/A 1.00 1.401.00 0.84 94 BF390024 Ncor1 1.00 1.34 0.93 0.89 92 BE098713 zzN/A 1.001.11 0.87 0.82 86 BF404414 zzN/A 1.00 1.19 0.85 0.80 82 AA945828 zzN/A1.00 1.54 0.84 1.07 88 AA849756 zzN/A 1.00 0.86 0.84 0.66 83 BM392373Ceacam1 1.00 1.38 0.82 0.87 78 BM385170 zzN/A 1.00 1.30 0.81 1.11 77BF411331 zzN/A 1.00 1.22 0.80 1.04 104 AI177513 zzN/A 1.00 0.90 0.800.70 107 AA964600 zzN/A 1.00 0.83 0.79 0.68 85 BI295141 zzN/A 1.00 1.070.78 0.82 108 AI227627 Cd9 1.00 0.74 0.77 0.61 90 NM_031762 Cdkn1b 1.001.02 0.74 0.45 89 AI180286 zzN/A 1.00 0.90 0.74 0.70 91 AI105205 Ctl11.00 0.93 0.73 0.57 103 BG380281 zzN/A 1.00 0.72 0.72 0.64 101 BI301490zzN/A 1.00 0.68 0.69 0.66 113 BM384203 zzN/A 1.00 0.90 0.69 0.66 114BG373555 zzN/A 1.00 0.98 0.68 0.74 105 BI288816 zzN/A 1.00 0.65 0.680.48 109 AA894262 zzN/A 1.00 1.07 0.68 0.59 106 AI228548 zzN/A 1.00 0.620.67 0.30 76 AW433971 Mvk 1.00 1.12 0.67 0.98 80 AA800750 zzN/A 1.001.13 0.65 0.92 84 AI171229 zzN/A 1.00 1.23 0.65 0.78 112 AI407835 Add31.00 0.74 0.65 0.65 97 NM_023989 LOC78973 1.00 0.84 0.65 0.70 81AA893602 zzN/A 1.00 1.13 0.65 0.93 98 AA943165 zzN/A 1.00 0.71 0.64 0.5293 NM_031347 Ppargc1 1.00 1.17 0.62 0.52 79 BE102350 zzN/A 1.00 1.640.61 1.18 110 AI408598 zzN/A 1.00 0.85 0.61 0.53 111 AI175820 zzN/A 1.000.64 0.61 0.57 99 AI179665 1.00 0.77 0.59 0.64 100 BI296591 zzN/A 1.000.53 0.54 0.55 102 AF189724 Cxcl12 1.00 0.73 0.50 0.61 96 BF417032 zzN/A1.00 0.87 0.49 0.57 95 M58040 Tfrc 1.00 0.63 0.45 0.42

TABLE 4 Genes differentially expressed in sensitized S1 DRG No.Identifier Acce. No. CTRL-VHL CTRL-CNI IBS-VHL IBS-CNI 11 1376554_atBE121079 1 1.74 2.23 2.01 23 1369233_at AF196965 1 1.45 2.09 1.71 41374620_at BM392373 1 1.43 1.87 2.41 6 1390403_at BE108405 1 1.39 1.831.86 8 1379272_at AA963084 1 1.08 1.83 1.70 24 1369157_at NM_017229 11.05 1.78 1.09 19 1382915_at AI237079 1 1.26 1.76 1.38 25 1374802_atAI010721 1 0.90 1.72 0.96 5 1389222_at BI282847 1 1.30 1.69 1.66 221368379_at NM_054001 1 1.31 1.68 1.66 7 1368678_at X67108 1 1.38 1.661.72 10 1386218_at AI639301 1 1.35 1.65 1.58 26 1370301_at U65656 1 1.171.64 1.02 17 1389099_at AI600184 1 1.50 1.64 1.50 18 1383159_at AW4344451 1.40 1.64 1.58 13 1384217_at BI276341 1 1.24 1.59 1.67 21 1389713_atAI602851 1 1.17 1.58 1.45 12 1372620_at AI008642 1 1.32 1.57 1.94 781385386_at BI302745 1 0.79 0.09 0.31 28 1387658_at U93849 1 1.60 0.430.82 47 1368703_at NM_053326 1 0.65 0.44 0.70 62 1377606_at AI500913 10.49 0.44 0.36 50 1375606_at AI235906 1 0.75 0.46 0.43 49 1369255_atNM_013123 1 0.80 0.46 0.58 40 1386999_at BG380730 1 0.78 0.47 0.68 421388589_at BG381046 1 0.74 0.47 0.61 41 1370666_at AF201839 1 0.66 0.490.44 52 1389864_at BF405086 1 0.69 0.50 0.58 72 1374283_at BF419505 10.82 0.51 0.59 34 1375469_at BE111847 1 0.67 0.52 0.67 38 1374002_atAI045904 1 0.71 0.55 0.79 65 1394114_at AA799434 1 0.67 0.56 0.63 671388101_at AF389425 1 0.73 0.56 0.71 57 1368279_at NM_053718 1 0.78 0.560.39 45 1376350_at BF396151 1 0.90 0.57 0.75 74 1376122_at BF401577 10.77 0.57 0.44 36 1369036_at NM_019309 1 0.72 0.58 0.65 66 1367814_atM14137 1 0.67 0.59 0.67 71 1372177_at AI180033 1 0.63 0.60 0.61 701373981_at BI299720 1 0.62 0.60 0.55 35 1383468_at BM958512 1 0.66 0.620.67 46 1376931_at BG380736 1 0.73 0.62 0.75 75 1371281_at M37568 1 0.970.63 0.39 44 1387818_at NM_053736 1 0.87 0.63 0.75 51 1369048_atNM_017289 1 0.71 0.64 0.56 37 1374084_at BE119993 1 0.68 0.64 0.59 301387327_at NM_133318 1 1.25 0.65 0.66 39 1373031_at BI275757 1 0.83 0.650.77 32 1375448_at BM391628 1 0.90 0.65 0.74 88 1369884_at NM_022182 10.90 0.66 0.54 59 1375294_at BF415950 1 0.75 0.66 0.42 90 1387404_atNM_078620 1 1.02 0.66 0.50 69 1386907_at NM_012949 1 0.75 0.67 0.67 631392500_at AA957990 1 0.59 0.67 0.58 68 1369000_at NM_021589 1 0.75 0.670.67

TABLE 5 Genes differentially expressed in CNI1493-treated colon No.Identifier Acce. No. Description 1 1370531_at U69550 phospholipase Dgene 1 2 1369708_at NM_031017 cAMP response element binding protein 1 31368889_at NM_023101 SNARE Vti1a-beta protein 4 1387046_at NM_053792selective LIM binding factor 5 1371192_at BF566236 neurofibromatosis 2 61369195_at NM_013068 Fatty acid binding protein 2 7 1398540_at BM386789Rattus norvegicus transcribed sequences 8 1375464_at BI290815 Rattusnorvegicus transcribed sequences 9 1387119_at AW433971 mevalonate kinase10 1374034_at BG379410 Rattus norvegicus transcribed sequence withstrong similarity to protein sp: P49589 (H. sapiens) SYC_HUMANCYSTEINYL-TRNA SYNTHETASE (CYSTEINE--TRNA LIGASE) (CYSRS) 11 1392633_atAI045724 Rattus norvegicus transcribed sequences 12 1371027_at BF556820Cas-Br-M (murine) ectropic retroviral transforming sequence b 131376708_at BM385170 Rattus norvegicus transcribed sequences 141387220_at NM_019323 mast cell protease 9 15 1377034_at BF411331 “Rattusnorvegicus transcribed sequence with moderate similarity to protein ref:NP_109591.1 (H. sapiens) serine (or cysteine) proteinase inhibitor,clade B (ovalbumin), member 1; protease inhibitor 2 (anti-elastase),monocyte/neutrophil; protease inhibitor 16 1374324_at AA945828 Rattusnorvegicus transcribed sequences 17 1370510_a_at AB012600 arylhydrocarbon receptor nuclear translocator-like 18 1387130_at NM_133315“solute carrier family 39 (iron-regulated transporter), member 1” 191390562_s_at BE102350 Rattus norvegicus transcribed sequences 201390960_at AA893602 Rattus norvegicus transcribed sequences 211371925_at AA893621 Rattus norvegicus clone C201 intestinal epitheliumproliferating cell-associated mRNA sequence 22 1370693_a_at M18630cyclic nucleotide phosphodiesterase 1 23 1377036_at BE102350 Rattusnorvegicus transcribed sequences 24 1392794_at AA893579 Rattusnorvegicus transcribed sequences 25 1388694_at AI233121 MHC class IRT1.O type 149 processed pseudogene 26 1388754_at AI176839 Rattusnorvegicus transcribed sequences 27 1368233_at NM_031042 “generaltranscription factor IIF, polypeptide 2 (30 kD subunit)” 28 1368679_a_atL14782 lyn protein non-receptor kinase 29 1372177_at AI180033 Rattusnorvegicus transcribed sequence with moderate similarity to protein ref:NP_004522.1 (H. sapiens) molybdenum cofactor synthesis 2 [Homo sapiens]30 1372868_at BF284295 Rattus norvegicus transcribed sequence with weaksimilarity to protein sp: O14657 (H. sapiens) TO1B_HUMAN Torsin Bprecursor 31 1380472_at AI639486 Rattus norvegicus transcribed sequencewith weak similarity to protein ref: NP_003860.1 (H. sapiens)carboxylesterase 2; intestinal carboxylesterase; livercarboxylesterase-2 [Homo sapiens] 32 1387994_at U89280 oxidative 17 betahydroxysteroid dehydrogenase type 6 33 1390531_at BE098021 Rattusnorvegicus transcribed sequences 34 1388055_at U39207 cytochrome P4504F5 35 1368379_at NM_054001 “CD36 antigen (collagen type I receptor,thrombospondin receptor)-like 2” 36 1368101_at NM_012518 calmodulin 3 371389302_at BI289482 Rattus norvegicus transcribed sequence with weaksimilarity to protein pir: S30833 (S. cerevisiae) S30833 hypotheticalprotein YEL044w - yeast (Saccharomyces cerevisiae) 38 1389378_atAI599324 Rattus norvegicus transcribed sequence with weak similarity toprotein pir: A42973 (H. sapiens) A42973 serum protein MSE55 - human 391387444_at NM_133592 brain-enriched membrane-associated protein tyrosineBEM-2 40 1374284_at AI227769 Rattus norvegicus transcribed sequences 411371239_s_at AF053361 “tropomyosin 3, gamma” 42 1387596_at NM_053897“Proteinase-activated receptor-2, G protein-coupled receptor 11” 431389873_at BI282953 apoptosis-associated speck-like protein containing aCARD 44 1369773_at NM_017105 Bone morphogenetic protein 3 45 1374119_atBI279615 “Rattus norvegicus transcribed sequence with strong similarityto protein ref: NP_004424.1 (H. sapiens) E74- like factor 3 (ets domaintranscription factor, epithelial- specific); E74-like factor 3 (etsdomain transcription factor); ets domain transcription 46 1368128_atNM_031598 “phospholipase A2, group IIA (platelets, synovial fluid)” 471368270_at NM_012907 Apolipoprotein B editing protein 48 1367960_atNM_019186 ADP-ribos lation-like 4 49 1367942_at NM_019144 acidphosphatase 5 50 1392694_at AW526101 Rattus norvegicus transcribedsequences 51 1374452_at BF399743 phosphodiesterase 9A 52 1368727_atNM_053929 “solute carrier family 7 (cationic amino acid transporter, y+system), member 9” 53 1376625_at BI296015 Rattus norvegicus transcribedsequences 54 1367768_at NM_031655 latexin 55 1369193_at AF474979 cyclindependent kinase inhibitor 2B 56 1382714_at AA875186 Rattus norvegicustranscribed sequences 57 1376359_at BG375355 “Rattus norvegicustranscribed sequence with weak similarity to protein ref: NP_113645.1(H. sapiens) membrane-spanning 4-domains, subfamily A, member 8B [Homosapiens]” 58 1372064_at BI296385 Rattus norvegicus CDK104 mRNA 591388396_at BI275932 Rattus norvegicus transcribed sequence with strongsimilarity to protein sp: O00506 (H. sapiens) ST25_HUMANSerine/threonine protein kinase 25 (Sterile 20/oxidant stress-responsekinase 1) (Ste20/oxidant stress response kinase-1) (SOK-1) (Ste20- likekinase) 60 1384191_at BF387765 Rattus norvegicus transcribed sequences61 1372255_at BF283284 Rattus norvegicus transcribed sequence withstrong similarity to protein sp: P54136 (H. sapiens) SYR_HUMANARGINYL-TRNA SYNTHETASE (ARGININE--TRNA LIGASE) (ARGRS) 62 1369262_atNM_022277 caspase-8 63 1376117_at BI289103 Rattus norvegicus transcribedsequence with moderate similarity to protein ref: NP_079533.1 (H.sapiens) NG22 protein; choline transporter-like protein 4 [Homo sapiens]64 1380262_at AA893436 Rattus norvegicus transcribed sequences 651370113_at NM_023987 inhibitor of apoptosis protein 1 66 1368437_atNM_019174 carbonic anhydrase 4 67 1390455_at AI013474 Rattus norvegicustranscribed sequence with strong similarity to protein sp: P08910 (H.sapiens) HPS1 HUMAN Protein PHPS1-2 68 1378658_at BI292185 Rattusnorvegicus transcribed sequence with weak similarity to protein ref:NP_036260.1 (H. sapiens) calcium activated chloride channel 4 [Homosapiens] 69 1376976_at AI009823 Rattus norvegicus transcribed sequencewith weak similarity to protein ref: NP_002995.1 (H. sapiens) secretedand transmembrane 1 precursor; K12 protein perecursor; type 1atransmembrane protein [Homo sapiens] 70 1370706_a_at U39943 cytochromeP450 monooxygenase 71 1372997_at AI105243 Rattus norvegicus transcribedsequences 72 1369183_at NM_019231 mitogen activated protein kinase 13 731386917_at NM_012744 Pyruvate carboxylase 74 1390678_at AA955527 Rattusnorvegicus transcribed sequences 75 1398847_at BG376935diphosphoinositol polyphosphate phosphohydolase type II 76 1368073_atNM_012591 Interferon regulatory factor 1 77 1371394_x_at BG664827“Rattus norvegicus endogenous retrovirus mRNA, partial sequence” 781370422_at AF036537 homocysteine respondent protein HCYP2 79 1375230_atAA800192 “Rattus norvegicus endogenous retrovirus mRNA, partialsequence” 80 1368007_at NM_022849 deleted in malignant brain tumors 1 811372671_at BI284293 Rattus norvegicus transcribed sequence with strongsimilarity to protein ref: NP_060809.1 (H. sapiens) hypothetical proteinFLJ11149 [Homo sapiens] 82 1368219_at NM_017137 chloride channel 2 831367760_at D13341 mitogen activated protein kinase kinase 1 841388006_at U89744 putative cell surface antigen 85 1367925_at NM_022715major vault protein 86 1390128_at BF557618 Rattus norvegicus transcribedsequence with strong similarity to protein ref: NP_065145.1 (H. sapiens)CHMP1.5 protein [Homo sapiens] 87 1388745_at AI228417 “Rattus norvegicustranscribed sequence with strong similarity to protein pdb: 1BGM (E.coli) O Chain O, Beta-Galactosidase (Chains I-P)” 88 1369940_atNM_031811 transaldolase 1 89 1370400_at L23128 “Rattus norvegicus MHCclass I mRNA, complete cds” 90 1371970_at AA799328 Rattus norvegicustranscribed sequences 91 1386933_at NM_134418 secretory (zymogen)granule membrane glycoprotein GP2 92 1369100_at NM_134375angiotensin/vasopressin receptor 93 1372619_at AI172185 “Rattusnorvegicus Aa2-277 mRNA, complete cds” 94 1380129_at AA818937 Rattusnorvegicus transcribed sequence with weak similarity to protein sp:P10266 (H. sapiens) POL1_HUMAN Endogenous retrovirus HERV-K10 putativepol polyprotein [Includes: Reverse transcriptase; Endonuclease] 951371078_at AI500830 RT1 class Ib gene 96 1367586_at NM_017025 lactatedehydrogenase A 97 1374033_at BG373505 Rattus norvegicus transcribedsequence with moderate similarity to protein pir: I38135 (H. sapiens)I38135 multicatalytic endopeptidase complex (EC 3.4.99.46) beta chainMECL-1 - human 98 1373913_at BF282271 Rattus norvegicus transcribedsequence with strong similarity to protein pir: T50626 (H. sapiens)T50626 hypothetical protein DKFZp762K1914.1 - human (fragment) 991372665_at AI230228 “Rattus norvegicus phosphoserine aminotransferasemRNA, complete cds” 100 1376056_at BF291214 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_116178.1 (H. sapiens) hypothetical protein FLJ14464 [Homo sapiens]101 1368066_at NM_053812 BCL2-antagonist/killer 1 102 1374838_atBI293504 Rattus norvegicus transcribed sequence with weak similarity toprotein sp: Q13342 (H. sapiens) LY10_HUMAN LYSP100 protein(Lymphoid-restricted homolog of Sp100) (Nuclear autoantigen Sp-140)(Speckled 140 kDa) (Nuclear body protein Sp140) 103 1372816_at BE107319Rattus norvegicus transcribed sequences 104 1388236_x_at M24026 RT1class Ib gene 105 1371210_s_at AJ276126 RT1 class Ib gene 106 1390325_atBI289418 Rattus norvegicus transcribed sequence with weak similarity toprotein ref: NP_083693.1 (M. musculus) RIKEN cDNA 9030605E16 [Musmusculus] 107 1369279_at NM_130819 retinol dehydrogenase homolog 1081390021_at BM391206 histone 2b 109 1368317_at NM_019157 aquaporin 7 1101387100_at NM_031703 aquaporin 3 111 1368975_at NM_013127 CD38 antigen112 1386908_at NM_022278 glutaredoxin 1 (thioltransferase) 1131369427_at NM_022617 macrophage expressed gene 1 114 1369110_x_atNM_012645 RT1 class Ib gene 115 1369957_at NM_019341 regulator ofG-protein signaling 5 116 1398985_at AI716480 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_004277.1 (H. sapiens) GTP binding protein 1 [Homo sapiens] 1171368413_at NM_022935 Amiloride binding protein 1 118 1370960_at BE104060insulin-like growth factor-binding protein 5 119 1368505_at NM_017214regulator of G-protein signaling 4 120 1373386_at AI179953 Rattusnorvegicus transcribed sequences 121 1387348_at BE113270 insulin-likegrowth factor-binding protein 5 122 1370638_at AF069525 ankyrin 3 (G)123 1377112_at AA859352 Rattus norvegicus transcribed sequence withmoderate similarity to protein ref: NP_001776.1 (H. sapiens) cytidinedeaminase [Homo sapiens] 124 1389611_at AA849857 Very low densitylipoprotein receptor 125 1369098_at NM_013155 Very low densitylipoprotein receptor 126 1368294_at NM_053907 deoxyribonuclease I-like 3127 1368342_at NM_031544 Adenosine monophosphate deaminase 3 1281368965_at NM_030834 monocarboxylate transporter 129 1392819_at AW921478“Rattus norvegicus transcribed sequence with weak similarity to proteinref: NP_071744.1 (H. sapiens) CD20- like precusor; membrane-spanning4-domains, subfamily A, member 6 [Homo sapiens]” 130 1367774_atNM_031509 “glutathione S-transferase, alpha 1” 131 1387687_at NM_133542“immunoglobulin superfamily, member 6” 132 1369173_at NM_032060complement component 3a receptor 1 133 1372013_at BG380285 Rattusnorvegicus transcribed sequence with weak similarity to protein ref:NP_006426.1 (H. sapiens) interferon induced transmembrane protein 2(1-8D); interferon-inducible [Homo sapiens] 134 1373025_at AI411618Rattus norvegicus transcribed sequence with moderate similarity toprotein pir: C1HUQC (H. sapiens) C1HUQC complement subcomponent C1qchain C precursor - human 135 1376652_at BF418957 Rattus norvegicustranscribed sequence with moderate similarity to protein pir: C1HUQA (H.sapiens) C1HUQA complement subcomponent C1q chain A precursor - human136 1368420_at NM_012532 ceruloplasmin 137 1373932_at BE098739 Rattusnorvegicus transcribed sequences 138 1387893_at D88250 “complementcomponent 1, s subcomponent” 139 1388557_at BF284922 Rattus norvegicustranscribed sequences 140 1374730_at AI102519 Rattus norvegicustranscribed sequence with weak similarity to protein ref: NP_003323.1(H. sapiens) TYRO protein tyrosine kinase binding protein; polycysticlipomembranous osteodysplasia with sclerosing leukoencephalopathy [Homosapiens] 141 1368000_at NM_016994 Complement component 3 1421368558_s_at NM_017196 allograft inflammatory factor 1 143 1373523_atAI011757 Rattus norvegicus transcribed sequence with weak similarity toprotein sp: P08637 (H. sapiens) FC3A_HUMAN Low affinity immunoglobulingamma FC region receptor III-A precursor (IGG FC receptor III- 2)(FC-gamma RIII-alpha) (FC-gamma RIIIA) (FCRIIIA) (FC-gamm 144 1387011_atNM_130741 lipocalin 2 145 1370885_at AA849399 cathepsin Y 146 1389470_atAI639117 Complement component 2 147 1368430_at AF154349 “protease,cysteine, 1 (legumain)” 148 1375010_at AI177761 Rattus norvegicustranscribed sequence with weak similarity to protein ref: NP_001242.1(H. sapiens) CD68 antigen; Macrophage antigen CD68 (microsialin) [Homosapiens] 149 1389006_at AI170394 Rattus norvegicus transcribed sequences150 1373575_at BE111722 Rattus norvegicus transcribed sequence withmoderate similarity to protein pir: A35241 (H. sapiens) A35241 IgE Fcreceptor gamma chain precursor - human 151 1390510_at BI294706 “Rattusnorvegicus transcribed sequence with weak similarity to protein ref:NP_071744.1 (H. sapiens) CD20- like precusor; membrane-spanning4-domains, subfamily A, member 6 [Homo sapiens]” 152 1376390_at BF395317“Rattus norvegicus transcribed sequence with weak similarity to proteinref: NP_071744.1 (H. sapiens) CD20- like precusor; membrane-spanning4-domains, subfamily A, member 6 [Homo sapiens]” 153 1368187_atNM_133298 glycoprotein (transmembrane) nmb 154 1389553_at BF393825“Rattus norvegicus transcribed sequence with weak similarity to proteinref: NP_057268.1 (H. sapiens) C-type (calcium dependent,carbohydrate-recognition domain) lectin, superfamily member 6; dendriticcell immunoreceptor; C-type lectin [Homo sapiens]” 155 1367568_a_atNM_012862 matrix Gla protein 156 1370628_at M34097 granzyme B 1571379604_at BF284937 “Rattus norvegicus transcribed sequence with weaksimilarity to protein ref: NP_085146.1 (H. sapiens) apolipoprotein L, 4[Homo sapiens]” 158 1368464_at NM_022393 macrophage galactoseN-acetyl-galactosamine specific lectin 159 1379766_at AI500952 Rattusnorvegicus transcribed sequences 160 1370516_at AB026665peptide/histidine transporter PHT2 161 1373071_at AI103101 Rattusnorvegicus transcribed sequence with strong similarity to protein pir:T00702 (H. sapiens) T00702 hypothetical protein F25965 1 - human(fragment) 162 1393224_at AW529774 Rattus norvegicus transcribedsequences 163 1390312_at BG670441 Rattus norvegicus transcribed sequencewith weak similarity to protein ref: NP_060124.1 (H. sapiens)hypothetical protein FLJ20073 [Homo sapiens] 164 1377412_at AI146237Rattus norvegicus transcribed sequences 165 1399125_at BI275516 Rattusnorvegicus transcribed sequence with weak similarity to protein sp:P49441 (H. sapiens) INPP_HUMAN Inositol polyphosphate 1-phosphatase(IPPase) (IPP) 166 1386986_at NM_053340 opioid growth factor receptor167 1374731_at BI275929 “Rattus norvegicus transcribed sequence withmoderate similarity to protein pdb: 1LBG (E. coli) B Chain B, LactoseOperon Repressor Bound To 21-Base Pair Symmetric Operator Dna, AlphaCarbons Only” 168 1388880_at BI278962 Lysosomal associated membraneprotein 1 (120 kDa) 169 1376075_at BM385544 Rattus norvegicustranscribed sequences 170 1369559_a_at NM_019195 integrin-associatedprotein 171 1376972_at AI407028 “solute carrier family 39(iron-regulated transporter), member 1” 172 1388776_at AI169176 Rattusnorvegicus transcribed sequence with weak similarity to protein ref:NP_057563.1 (H. sapiens) hypothetical protein LOC51246 [Homo sapiens]173 1376029_at BI295991 Rattus norvegicus transcribed sequences 1741388164_at AF029241 “Rat MHC class I RT1.C/E mRNA, 3′ end” 1751389734_x_at BI282965 RT1 class Ib gene 176 1370429_at L40362 RT1 classIb gene 177 1388900_at BG381414 Rattus norvegicus transcribed sequences178 1375955_at BI289415 Rattus norvegicus transcribed sequences 1791389011_at BG374333 Rattus norvegicus transcribed sequences 1801389387_at BF561377 hydroxyindole-O-methyltransferase 181 1387206_atNM_031740 “UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase,polypeptide 6” 182 1388071_x_at M24024 RT1 class Ib gene 183 1398925_atBI274664 Rattus norvegicus transcribed sequence with weak similarity toprotein ref: NP_060818.1 (H. sapiens) hypothetical protein FLJ11171[Homo sapiens] 184 1368224_at NM_031531 Serine protease inhibitor 1851374718_at AA945915 Rattus norvegicus transcribed sequences 1861394340_at BF523172 Rattus norvegicus transcribed sequence with weaksimilarity to protein sp: P49441 (H. sapiens) INPP_HUMAN Inositolpolyphosphate 1-phosphatase (IPPase) (IPP) 187 1376022_at BI292196Rattus norvegicus transcribed sequences 188 1368732_at NM_032056“transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)” 1891367710_at NM_017257 “protease (prosome, macropain) 28 subunit, beta”190 1367786_at NM_080767 “proteasome (prosome, macropain) subunit, betatype, 8 (low molecular mass polypeptide 7)” 191 1389170_at BF283754Rattus norvegicus transcribed sequences 192 1375853_at BF284358 Rattusnorvegicus transcribed sequences 193 1373757_at AW529298 Rattusnorvegicus transcribed sequence with moderate similarity to protein ref:NP_006691.1 (H. sapiens) FLN29 gene product [Homo sapiens] 1941388149_at X57523 “transporter 1, ATP-binding cassette, sub-family B(MDR/TAP)” 195 1372604_at BI289459 “Rattus norvegicus transcribedsequence with weak similarity to protein ref: NP_055164.1 (H. sapiens)apolipoprotein L, 3; TNF-inducible protein CG12-1 [Homo sapiens]” 1961387027_a_at U72741 “Lectin, galactose binding, soluble 9 (Galectin-9)”197 1372034_at BF284106 Rattus norvegicus transcribed sequences 1981370186_at AI599350 “proteosome (prosome, macropain) subunit, beta type9 (large multifunctional protease 2)” 199 1388212_a_at AJ243974 “Rat MHCclass I RT1.C/E mRNA, 3′ end” 200 1388213_a_at AJ243973 “Rat MHC class IRT1.C/E mRNA, 3′ end” 201 1371123_x_at AJ243973 “Rat MHC class I RT1.C/EmRNA, 3′ end” 202 1371152_a_at Z18877 25 oligoadenylate synthetase 2031372930_at AI411381 “Rattus norvegicus cDNA, clone: aC10, differentiallyexpressed in pylorus” 204 1388791_at BI275911 Rattus norvegicustranscribed sequence with moderate similarity to protein pir: T14738 (H.sapiens) T14738 hypothetical protein DKFZp564A2416.1 - human (fragment)205 1369716_s_at NM_012976 “Lectin, galactose binding, soluble 5(Galectin-5)” 206 1376496_at AI717736 “Rattus norvegicus transcribedsequence with weak similarity to protein ref: NP_112092.1 (H. sapiens)apolipoprotein L, 2 [Homo sapiens]” 207 1374337_at AI408954 “Rattusnorvegicus transcribed sequence with moderate similarity to protein pdb:1LBG (E. coli) B Chain B, Lactose Operon Repressor Bound To 21-Base PairSymmetric Operator Dna, Alpha Carbons Only” 208 1387242_at NM_019335“Protein kinase, interferon-inducible double stranded RNA dependent” 2091376144_at AA819679 Rattus norvegicus transcribed sequence with weaksimilarity to protein ref: NP_113646.1 (H. sapiens) B aggressivelymphoma gene [Homo sapiens] 210 1368835_at AW434718 signal transducerand activator of transcription 1 211 1387946_at AF065438 peptidylprolylisomerase C-associated protein 212 1383564_at BF411036 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_004022.1 (H. sapiens) interferon regulatory factor 7 isoform d [Homosapiens] 213 1389034_at BI295179 Rattus norvegicus transcribed sequencewith weak similarity to protein sp: Q9UMW8 (H. sapiens) UBPI_HUMANUbiquitin carboxyl-terminal hydrolase 18 (Ubiquitin thiolesterase 18)(Ubiquitin-specific processing protease 18) (Deubiquitinating enzyme 18)(Ubiqui 214 1388347_at AI233210 Rattus norvegicus transcribed sequencewith weak similarity to protein sp: Q16553 (H. sapiens) LY6E_HUMANLymphocyte antigen Ly-6E precursor (Retinoic acid-induced gene Eprotein) (RIG-E) (Thymic shared antigen-1) (TSA-1) (Stem cell antigen 2)(SCA-2) 215 1376845_at AA819034 Rattus norvegicus transcribed sequencewith moderate similarity to protein ref: NP_114425.1 (H. sapiens) TLH29protein precursor [Homo sapiens] 216 1376693_at AA998964 Rattusnorvegicus transcribed sequence with moderate similarity to protein ref:NP_060124.1 (H. sapiens) hypothetical protein FLJ20073 [Homo sapiens]217 1387770_at NM_130743 “interferon, alpha-inducible protein 27-like”218 1376920_at BF408536 Rattus norvegicus transcribed sequence with weaksimilarity to protein ref: NP_060124.1 (H. sapiens) hypothetical proteinFLJ20073 [Homo sapiens] 219 1387354_at NM_032612 signal transducer andactivator of transcription 1 220 1373037_at BI279216 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_004214.1 (H. sapiens) ubiquitin-conjugating enzyme E2L 6 [Homosapiens] 221 1387995_a_at BI285494 interferon induced transmembraneprotein 3-like 222 1377497_at BF419319 “Rattus norvegicus transcribedsequence with moderate similarity to protein pir: I60307 (E. coli)I60307 beta- galactosidase, alpha peptide - Escherichia coli” 2231368227_at NM_031664 “solute carrier family 28, member 2” 224 1390507_atBI296097 Rattus norvegicus transcribed sequence with moderate similarityto protein ref: NP_002192.2 (H. sapiens) interferon stimulated gene (20kD) [Homo sapiens] 225 1371440_at AW916647 Prostaglandin F receptor 2261369590_a_at NM_024134 DNA-damage inducible transcript 3 227 1369202_atNM_017028 myxovirus (influenza virus) resistance 2 228 1374627_atBF283727 Rattus norvegicus transcribed sequences 229 1374551_at BM388891Rattus norvegicus transcribed sequence with weak similarity to proteinpir: JC5262 (H. sapiens) JC5262 leucine zipper protein IFP35 - human 2301387134_at NM_053687 schlafen 4 231 1373514_at AA899109 Rattusnorvegicus transcribed sequences 232 1372585_at BM388445 Rattusnorvegicus transcribed sequences 233 1369031_at NM_053374 interferongamma inducing factor binding protein 234 1371070_at AJ302054 tumorstroma and activated macrophage protein DLM-1 235 1367595_s_at NM_012512Beta-2-microglobulin 236 1367663_at NM_017264 “protease (prosome,macropain) 28 subunit, alpha” 237 1390042_at AI071166 Rattus norvegicustranscribed sequences 238 1369186_at D85899 caspase 1 239 1389571_atBG666368 Rattus norvegicus transcribed sequence with moderate similarityto protein sp: P52630 (H. sapiens) STA2_HUMAN Signal transducer andactivator of transcription 2 (p113) 240 1370913_at AI409634 Best5protein 241 1387969_at U22520 chemokine (C-X-C motif) ligand 10 2421376908_at AW531805 Rattus norvegicus transcribed sequence with weaksimilarity to protein sp: O14879 (H. sapiens) IFT4_HUMANInterferon-induced protein with tetratricopeptide repeats 4 (IFIT-4)(Interferon-induced 60 kDa protein) (IFI-60K) (ISG-60) (CIG49) (Retinoicacid-ind 243 1369973_at NM_017154 xanthine dehydrogenase 244 1368332_atNM_133624 “guanylate binding protein 2, interferon-inducible” 2451372254_at AW915763 Rattus norvegicus transcribed sequence with moderatesimilarity to protein sp: P05155 (H. sapiens) IC1_HUMAN Plasma proteaseC1 inhibitor precursor (C1 Inh) (C1Inh) 246 1373197_at AI578263 Rattusnorvegicus transcribed sequences 247 1370892_at BI285347 complementcomponent 4a 248 1372757_at BM386875 signal transducer and activator oftranscription 1 249 1376151_a_at AI407953 Rattus norvegicus transcribedsequences 250 1387283_at NM_134350 myxovirus (influenza virus)resistance 2 251 1371015_at X52711 myxovirus (influenza virus)resistance 252 1373992_at AI408440 “Rattus norvegicus cDNA, clone: aD9,differentially expressed in pylorus” 253 1390738_at BM385476 Rattusnorvegicus mRNA for DAMP-1 protein 254 1388056_at AF068268 2′5′oligoadenylate synthetase-2 255 1389365_at AI228291 Rattus norvegicustranscribed sequences 256 1369726_at NM_033098 TAP binding protein 2571370172_at AA892254 superoxide dismutase 2 258 1386893_at NM_017113granulin 259 1375796_at BI300770 “Rattus norvegicus Ac2-233 mRNA,complete cds” 260 1389014_at BI297612 pre-B-cell colony-enhancing factor261 1371209_at AJ243338 RT1 class Ib gene 262 1388255_x_at AJ243338 RT1class Ib gene 263 1373406_at BM384991 Rattus norvegicus transcribedsequences 264 1375006_at BE121050 Rattus norvegicus transcribedsequences 265 1387897_at L16532 cyclic nucleotide phosphodiesterase 1266 1369456_at NM_017250 5-hydroxytryptamine (serotonin) receptor 2B 2671374141_at BG372419 Rattus norvegicus transcribed sequence with strongsimilarity to protein ref: NP_071387.1 (H. sapiens) chromosome 20 openreading frame 67 [Homo sapiens] 268 1374678_at BE109578 Rattusnorvegicus transcribed sequences 269 1372968_at BM385950 Rattusnorvegicus transcribed sequence with weak similarity to protein pir:S37032 (R. norvegicus) S37032 gene LL5 protein - rat 270 1371832_atAW526333 Rattus norvegicus transcribed sequences 271 1390117_at BG372455Rattus norvegicus transcribed sequences 272 1369381_a_at D50306 “solutecarrier family 15 (oligopeptide transporter), member 1” 273 1388103_atAF361355 voltage-dependent calcium channel gamma subunit-like protein274 1386943_at NM_022533 plasmolipin 275 1373060_at AI406281 Rattusnorvegicus transcribed sequence with strong similarity to protein pir:T12468 (H. sapiens) T12468 hypothetical protein DKFZp564O123.1 - human276 1375911_at AI171772 “Rattus norvegicus hypothetical protein LK44mRNA, complete cds” 277 1370071_at NM_130399 Adenosine deaminase 2781368762_at NM_053299 ubiquitin D 279 1369712_at NM_031735serine/threonine kinase 3 280 1368419_at AF202115 ceruloplasmin 2811369029_at NM_057194 phospholipid scramblase 1 282 1369888_at NM_012707glucagon 283 1398879_at BE329031 arrestin-E 284 1369942_at NM_031675actinin alpha 4 285 1370807_at AF411216 vacuole Membrane Protein 1 2861377124_at AA964824 Rattus norvegicus transcribed sequence with weaksimilarity to protein pir: S48059 (H. sapiens) S48059 metal-regulatorytranscription factor - human 287 1390221_at BM385216 Rattus norvegicustranscribed sequences 288 1390604_s_at BM387863 Rattus norvegicustranscribed sequence with weak similarity to protein ref: NP_055103.1(H. sapiens) integrin beta 3 binding protein (beta3-endonexin); beta 3endonexin [Homo sapiens] 289 1398916_at AI104388 heat shock 27 kDaprotein 1 290 1374600_at BM986536 germinal histone H4 gene 2911370307_at M64780 Agrin 292 1371888_at AA892843 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_078816.1 (H. sapiens) hypothetical protein FLJ20917 [Homo sapiens]293 1373603_at BG673166 Rattus norvegicus transcribed sequence withmoderate similarity to protein pir: T45061 (H. sapiens) T45061hypothetical protein c316G12.2 [imported]- human 294 1388408_at AA800199Rattus norvegicus transcribed sequence with weak similarity to proteinpir: T34520 (H. sapiens) T34520 hypothetical protein DKFZp564J157.1 -human (fragment) 295 1368471_at NM_013118 guanylate cyclase activator 2A296 1374401_at AI549249 Rattus norvegicus transcribed sequence withstrong similarity to protein sp: O60749 (H. sapiens) SNX2_HUMAN Sortingnexin 2 297 1371267_at M64795 zzN/A 298 1370832_at U06434 smallinducible cytokine A4 299 1383241_at BI292425 Rattus norvegicustranscribed sequence with moderate similarity to protein sp: P00736 (H.sapiens) C1R_HUMAN Complement C1r component precursor 300 1387922_atAF109674 late gestation lung protein 1 301 1376447_at AI706850 Rattusnorvegicus transcribed sequence with weak similarity to protein sp:P10163 (H. sapiens) PRB4_HUMAN Salivary proline-rich protein POprecursor (Allele S) 302 1367740_at M14400 “creatine kinase, brain” 3031387036_at NM_024360 hairy and enhancer of split 1 (Drosophila) 3041370360_at AF146738 testis specific protein 305 1389111_at BF396316Rattus norvegicus transcribed sequences 306 1374454_at BM388557 Rattusnorvegicus transcribed sequences 307 1367764_at NM_012923 Cyclin G1 3081368038_at AF260258 synaptojanin 2 binding protein 309 1372635_atBF282163 Rattus norvegicus transcribed sequences 310 1373571_at AI170276Rattus norvegicus transcribed sequences 311 1375872_at AI144944 Rattusnorvegicus transcribed sequences 312 1383767_at AW524430 Rattusnorvegicus transcribed sequences 313 1376325_at BM388975 Rattusnorvegicus transcribed sequence with strong similarity to protein pir:S52863 (H. sapiens) S52863 DNA-binding protein R kappa B - human 3141388485_at BG380414 Rattus norvegicus transcribed sequences 3151368782_at NM_019348 somatostatin receptor 2 316 1375867_at AW524493Rattus norvegicus transcribed sequences 317 1375138_at AA893169 Tissueinhibitor of metalloproteinase 3 318 1390112_at BF284634 “Rattusnorvegicus transcribed sequence with strong similarity to protein ref:NP_004096.2 (H. sapiens) EGF- containing fibulin-like extracellularmatrix protein 1 precursor, isoform a precursor; fibrillin-like [Homosapiens]” 319 1372935_at AI598550 Rattus norvegicus transcribedsequences 320 1368322_at NM_012880 superoxide dismutase 3 321 1372104_atBF289002 Rattus norvegicus transcribed sequence with weak similarity toprotein ref: NP_003106.1 (H. sapiens) UDP- N-acteylglucosaminepyrophosphorylase 1; AgX; sperm associated antigen 2;UDP-N-acteylglucosamine pyrophosphorylase 1; Sperm associated antigen 2[Hom 322 1367687_a_at M25719 Peptidylglycine alpha-amidatingmonooxygenase 323 1372455_at AI410264 Rattus norvegicus transcribedsequence with strong similarity to protein sp: O95859 (H. sapiens)TNE2_HUMAN Tetraspan NET-2 324 1372940_at BM389329 Rattus norvegicustranscribed sequences 325 1367631_at NM_022266 connective tissue growthfactor 326 1376344_at AI010267 Rattus norvegicus transcribed sequences327 1370408_at AF313411 putative small membrane protein NID67 3281390283_at BI274636 Rattus norvegicus transcribed sequences 3291372327_at BF552877 Rattus norvegicus transcribed sequence with strongsimilarity to protein ref: NP_057216.1 (H. sapiens) myelin geneexpression factor 2 [Homo sapiens] 330 1376175_at BF283433 Rattusnorvegicus transcribed sequences 331 1373966_at BF406242 Rattusnorvegicus transcribed sequence with moderate similarity to protein pir:T43490 (H. sapiens) T43490 hypothetical protein DKFZp434A139.1 - human(fragments) 332 1376749_at AA945955 Rattus norvegicus transcribedsequence with moderate similarity to protein pir: B35272 (H. sapiens)B35272 osteoinductive factor - human 333 1367604_at NM_022501cysteine-rich protein 2 334 1398345_at BM389225 angiopoietin-like 2 3351389836_a_at AI599265 Tissue inhibitor of metalloproteinase 3 3361389256_at BG381256 Rattus norvegicus transcribed sequences 3371371500_at BG375362 Rattus norvegicus transcribed sequence with moderatesimilarity to protein ref: NP_003564.1 (H. sapiens) latent transforminggrowth factor beta binding protein 4 [Homo sapiens] 338 1371629_atAI105444 “Rattus norvegicus transcribed sequence with moderatesimilarity to protein pir: I60307 (E. coli) I60307 beta- galactosidase,alpha peptide - Escherichia coli” 339 1387707_at NM_017102 “solutecarrier family 2, member 2” 340 1387505_at NM_013145 “Guanine nucleotidebinding protein, alpha inhibiting 1” 341 1388890_at BM390316 Rattusnorvegicus transcribed sequences 342 1373506_at AA944568 Rattusnorvegicus transcribed sequences 343 1369415_at NM_053328 “basichelix-loop-helix domain containing, class B2” 344 1372264_at BI277460“Rattus norvegicus transcribed sequence with strong similarity toprotein sp: P35558 (H. sapiens) PPCC_HUMAN Phosphoenolpyruvatecarboxykinase, cytosolic [GTP] (Phosphoenolpyruvate carboxylase)(PEPCK-C)” 345 1374105_at H31665 Rattus norvegicus transcribed sequences346 1387156_at NM_024391 17-beta hydroxysteroid dehydrogenase type 2 3471376569_at BM385790 Rattus norvegicus transcribed sequence with strongsimilarity to protein sp: Q9Y5W3 (H. sapiens) KLF2_HUMAN Kruppel-likefactor 2 (Lung kruppel-like factor) 348 1387028_a_at M86708 “Inhibitorof DNA binding 1, helix-loop-helix protein (splice variation)” 3491376661_at AI556122 Rattus norvegicus transcribed sequences 3501377287_at AA957673 Rattus norvegicus transcribed sequences 3511375910_at AA874943 Rattus norvegicus transcribed sequence with moderatesimilarity to protein pir: T46465 (H. sapiens) T46465 hypotheticalprotein DKFZp434A0530.1 - human 352 1388447_at AA800701 Rattusnorvegicus transcribed sequences 353 1370912_at BI278231 R. norvegicushsp70.2 mRNA for heat shock protein 70 354 1368883_at NM_030868 NOVprotein 355 1388945_at BM385779 “Rattus norvegicus transcribed sequencewith moderate similarity to protein pir: I60307 (E. coli) I60307 beta-galactosidase, alpha peptide - Escherichia coli” 356 1389514_at AI711152Rattus norvegicus transcribed sequences 357 1377086_at AI233530 Rattusnorvegicus transcribed sequences 358 1373947_at BI278545 Rattusnorvegicus transcribed sequence with strong similarity to protein pir:A47220 (H. sapiens) A47220 dermatopontin precursor - human 3591374171_at AI170507 “ATP-binding cassette, sub-family C (CFTR/MRP),member 9” 360 1372490_at BF283759 Rattus norvegicus transcribedsequences 361 1390257_at BG376956 Rattus norvegicus transcribedsequences 362 1369651_at NM_012673 Thymus cell surface antigen 3631388866_at AA799392 “Rattus norvegicus transcribed sequence withmoderate similarity to protein pdb: 1LBG (E. coli) B Chain B, LactoseOperon Repressor Bound To 21-Base Pair Symmetric Operator Dna, AlphaCarbons Only” 364 1368303_at NM_031678 period homolog 2 365 1370399_atM29853 “cytochrome P450, subfamily 4B, polypeptide 1” 366 1385606_atBF559626 Rattus norvegicus transcribed sequences 367 1376170_at BI290821Rattus norvegicus transcribed sequence with moderate similarity toprotein pir: S46657 (H. sapiens) S46657 collagen alpha 1(XIV) chain -human (fragments) 368 1376832_at AA800763 Rattus norvegicus transcribedsequences 369 1376049_at AA925805 Rattus norvegicus transcribedsequences 370 1398365_at AI171466 Rattus norvegicus transcribed sequencewith strong similarity to protein ref: NP_057048.1 (H. sapiens) CGI- 38protein [Homo sapiens] 371 1372847_at AW524458 Rattus norvegicustranscribed sequence with strong similarity to protein ref: NP_064571.1(H. sapiens) DC11 protein [Homo sapiens] 372 1372872_at BI291271 Rattusnorvegicus transcribed sequences 373 1376177_at AI179609 Rattusnorvegicus transcribed sequences 374 1387039_at NM_030828 glypican 1 3751372208_at AA942959 “protein phosphatase 1, regulatory (inhibitor)subunit 1B” 376 1376933_at AI170377 Rattus norvegicus transcribedsequences 377 1388034_at AB070355 kinesin family member 1B 3781371794_at BM391449 Rattus norvegicus transcribed sequence with moderatesimilarity to protein pir: T14273 (M. musculus) T14273 zinc fingerprotein 106 - mouse 379 1393464_at BM383378 Rattus norvegicustranscribed sequences 380 1389007_at AI231799 Rattus norvegicustranscribed sequences 381 1371508_at AI412098 “protein tyrosinephosphatase type IVA, member 2” 382 1374694_at BF284602 Rattusnorvegicus transcribed sequences 383 1374558_at AI010316 Rattusnorvegicus transcribed sequences 384 1399028_at AI171954 Rattusnorvegicus transcribed sequences 385 1368550_at NM_022858 HNF-3/forkheadhomolog-1 386 1370211_at BE106940 neurogranin 387 1387090_a_at NM_024135LIM motif-containing protein kinase 2 388 1370072_at NM_012608 membranemetallo endopeptidase 389 1371703_at AI407114 Complement component 3 3901390319_at BG378301 Rattus norvegicus transcribed sequence 3911387008_at NM_022948 tricarboxylate carrier-like protein 392 1389561_atBE110624 Rattus norvegicus transcribed sequence with strong similarityto protein ref: NP_079740.1 (M. musculus) RIKEN cDNA 1810021J13 [Musmusculus] 393 1376023_at BM386555 Rattus norvegicus transcribedsequences 394 1386979_at NM_133395 developmentally regulated proteinTPO1 395 1368163_at J02997 Dipeptidyl peptidase 4 396 1374935_atAI412099 “Rattus norvegicus transcribed sequence with moderatesimilarity to protein pir: I60307 (E. coli) I60307 beta- galactosidase,alpha peptide - Escherichia coli” 397 1387084_at NM_012789 Dipeptidylpeptidase 4 398 1369770_at NM_012719 somatostatin receptor 1 3991389601_at BI293610 “Rattus norvegicus transcribed sequence withmoderate similarity to protein pir: I60307 (E. coli) I60307 beta-galactosidase, alpha peptide - Escherichia coli” 400 1368295_atNM_080786 “solute carrier family 21 (organic anion transporter), member9” 401 1376242_at BF402365 Rattus norvegicus transcribed sequences 4021387059_at NM_019362 “serine threonine kinase 39 (STE20/SPS1 homolog,yeast)” 403 1389310_at BF400694 Rattus norvegicus transcribed sequences404 1387169_at NM_053400 “transducin-like enhancer of split 3, homologof Drosophila” 405 1387023_at NM_031154 “glutathione S-transferase, mutype 3 (Yb3)” 406 1398430_at AW524711 Rattus norvegicus transcribedsequences 407 1369249_at NM_053714 progressive ankylosis 408 1387913_atU48220 cytochrome P450 2D18 409 1370320_at AY083160 MAWD binding protein410 1376047_at BI285321 Rattus norvegicus transcribed sequences 4111387315_at NM_012878 “surfactant, pulmonary-associated protein D” 4121387239_a_at AB008803 “peptidyl arginine deiminase, type 4” 4131371477_at BG380735 Rattus norvegicus transcribed sequences 4141386974_at NM_134397 LL5 protein 415 1370930_at BF417285 kinesin 1C 4161371689_at BE107334 eukaryotic translation elongation factor 1 alpha 1417 1389040_at AI170825 Rattus norvegicus transcribed sequences 4181390710_x_at AA850618 Rattus norvegicus transcribed sequence with strongsimilarity to protein ref: NP_003096.1 (H. sapiens) sortilin-relatedreceptor containing LDLR class A repeats preproprotein; sortingprotein-related receptor containing LDLR class A repeats; low-density li419 1367700_at NM_080698 fibromodulin 420 1388317_at BE110655 Rattusnorvegicus transcribed sequences 421 1387184_at NM_024355 axin 2 4221390399_at BE102391 Rattus norvegicus transcribed sequences 4231375590_at AA894335 Rattus norvegicus transcribed sequence with moderatesimilarity to protein pir: T00368 (H. sapiens) T00368 hypotheticalprotein KIAA0663 - human 424 1387901_at L19180 “protein tyrosinephosphatase, receptor type, D” 425 1388768_at BI285601 Rattus norvegicustranscribed sequences 426 1373434_at AA944179 Rattus norvegicustranscribed sequences 427 1376784_at BI274481 Rattus norvegicustranscribed sequences 428 1390311_at AW528602 Rattus norvegicustranscribed sequences 429 1389150_at AW524559 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_076933.1 (H. sapiens) hypothetical protein MGC3265 [Homo sapiens] 4301372728_at BE103745 Rattus norvegicus transcribed sequence with moderatesimilarity to protein sp: Q99523 (H. sapiens) SORT_HUMAN Sortilinprecursor (Glycoprotein 95) (Gp95) (Neurotensin receptor 3) (NT3) (100kDa NT receptor) 431 1377325_a_at AW531278 Rattus norvegicus transcribedsequences 432 1389288_at BI279838 “Rattus norvegicus transcribedsequence with moderate similarity to protein ref: NP_002479.1 (H.sapiens) NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2 (8 kD,B8) [Homo sapiens]” 433 1375951_at AA818521 thrombomodulin 4341367880_at NM_012974 Laminin chain beta 2 435 1399109_at BI281673 Rattusnorvegicus transcribed sequences 436 1369926_at NM_022525 glutathioneperoxidase 3 437 1388145_at BM390128 Tenascin X 438 1398973_at BI296499Rattus norvegicus transcribed sequence with strong similarity to proteinsp: Q9HD45 (H. sapiens) T9S3_HUMAN Transmembrane 9 superfamily proteinmember 3 precursor (SM-11044 binding protein) (EP70- P-iso) 4391398990_at BI281754 Rattus norvegicus transcribed sequences 4401371245_a_at BI287300 hemoglobin beta chain complex 441 1392890_atBG663460 Rattus norvegicus transcribed sequences 442 1371102_x_at X05080hemoglobin beta chain complex 443 1370240_x_at AI179404 “hemoglobin,alpha 1” 444 1367553_x_at NM_033234 hemoglobin beta chain complex 4451370239_at AI179404 “hemoglobin, alpha 1” 446 1388608_x_at AI577319“hemoglobin, alpha 1” 447 1388848_at AA891760 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_071934.1 (H. sapiens) hypothetical protein FLJ22056 [Homo sapiens]448 1389186_at AA944175 “Rattus norvegicus transcribed sequence withmoderate similarity to protein pir: I60307 (E. coli) I60307 beta-galactosidase, alpha peptide - Escherichia coli” 449 1371972_at BM388888Rattus norvegicus transcribed sequence with moderate similarity toprotein pir: T00371 (H. sapiens) T00371 hypothetical protein KIAA0668 -human (fragment) 450 1372640_at BI277758 Rattus norvegicus transcribedsequence with moderate similarity to protein ref: NP_076223.1 (M.musculus) RIKEN cDNA 1200009H11 [Mus musculus] 451 1373162_at AI600085“Rattus norvegicus NYGGF3 mRNA, partial cds” 452 1387310_at NM_134462putative secretory pathway Ca-ATPase SPCA2 453 1388698_at AI407838extracellular matrix protein 1 454 1376105_at AI599143 Rattus norvegicustranscribed sequence with moderate similarity to protein pir: S46657 (H.sapiens) S46657 collagen alpha 1(XIV) chain - human (fragments) 4551373674_at BI283094 Rattus norvegicus transcribed sequence with moderatesimilarity to protein sp: Q13361 (H. sapiens) MGP2_HUMANMicrofibril-associated glycoprotein 2 precursor (MAGP-2) (MP25) 4561373952_at AI409841 Rattus norvegicus transcribed sequences 4571387625_at NM_013104 zzN/A 458 1374674_at AW528792 Rattus norvegicustranscribed sequences 459 1375844_at AI406370 Rattus norvegicustranscribed sequence with strong similarity to protein ref: NP_067013.1(H. sapiens) polypyrimidine tract binding protein 2; neuralpolypyrimidine tract binding protein; PTB-like protein [Homo sapiens]460 1368407_at NM_022605 heparanase 461 1377076_at AI716131 Rattusnorvegicus transcribed sequences 462 1375412_at AI101331 Rattusnorvegicus transcribed sequences 463 1376398_at BF417784 Rattusnorvegicus transcribed sequences 464 1399054_at BG375798 Rattusnorvegicus transcribed sequences 465 1374126_at BG374261 Rattusnorvegicus transcribed sequences 466 1368099_at NM_053722CLIP-associating protein 2 467 1387182_at NM_057201 G protein-coupledreceptor 37 (endothelin receptor type B-like) 468 1372814_at BF283084Rattus norvegicus transcribed sequences 469 1376115_at AA964244 Rattusnorvegicus transcribed sequences 470 1369373_at NM_053429 fibroblastgrowth factor receptor 3 471 1376781_at BI286116 Rattus norvegicustranscribed sequences 472 1369638_at NM_012947 Eukaryotic elongationfactor 2 kinase 473 1389003_at BI282008 Rattus norvegicus transcribedsequences 474 1376089_at BI294974 Low density lipoprotein receptor 4751367932_at NM_017268 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1476 1387809_at NM_053703 mitogen-activated protein kinase kinase 6 4771375549_at AI407719 Rattus norvegicus transcribed sequences 4781370209_at BE101336 Kruppel-like factor 9 479 1389311_at AW915161 Rattusnorvegicus transcribed sequences 480 1390164_at BE099850 Rattusnorvegicus transcribed sequences 481 1376989_at BE099568 Rattusnorvegicus transcribed sequences 482 1368656_at NM_053856 secretograninIII 483 1373415_at AI407050 Rattus norvegicus transcribed sequences 4841374819_at AI599945 Rattus norvegicus transcribed sequences 4851367594_at NM_017087 biglycan 486 1372624_at BF551377 Rattus norvegicustranscribed sequences 487 1376425_at BF420705 “transforming growthfactor, beta 2” 488 1389138_at AA945574 Rattus norvegicus transcribedsequences 489 1367940_at NM_053352 chemokine orphan receptor 1 4901371747_at AI406304 Rattus norvegicus transcribed sequence with moderatesimilarity to protein ref: NP_077275.1 (H. sapiens) chromosome 20 openreading frame 149 [Homo sapiens] 491 1372978_at BI291218 Rattusnorvegicus transcribed sequences 492 1387812_at NM_012999 Subtilisin -like endoprotease 493 1389794_at AI044984 Rattus norvegicus transcribedsequences 494 1388384_at AI407618 Rattus norvegicus transcribedsequences 495 1374557_at BF394235 Rattus norvegicus transcribed sequencewith strong similarity to protein ref: NP_079187.1 (H. sapiens)hypothetical protein FLJ23091 [Homo sapiens] 496 1370579_at U53513“glycine-, glutamate-, thienylcyclohexylpiperidine- binding protein” 4971367918_at NM_031066 protein kinase C-binding protein Zeta1 4981368930_at NM_023021 intermediate conductance calcium-activatedpotassium channel 499 1387654_at NM_023092 unconventional myosin Myr2 Iheavy chain 500 1367759_at NM_012578 Histone H1-0 501 1386957_atNM_053622 nuclear pore membrane glycoprotein 121 kD 502 1388709_atBF284695 Rattus norvegicus transcribed sequence with weak similarity toprotein pir: A35804 (H. sapiens) A35804 nucleolin - human 503 1384371_atAW921429 zzN/A 504 1371940_at AW920000 Rattus norvegicus transcribedsequence with strong similarity to protein sp: Q9UPN3 (H. sapiens)ACF7_HUMAN Actin cross-linking family protein 7 (Macrophin)(Trabeculin-alpha) (620 kDa actin-binding protein) (ABP620) 5051370131_at NM_031556 caveolin 506 1372825_at BI290551 Rattus norvegicustranscribed sequences 507 1367989_at NM_012751 “solute carrier family 2,member 4” 508 1376724_at AI170671 Rattus norvegicus transcribedsequences 509 1372967_at BI280323 Rattus norvegicus transcribedsequences 510 1370291_at AF002281 actinin alpha 2 associated LIM protein511 1377281_at BM388545 Rattus norvegicus transcribed sequence withmoderate similarity to protein ref: NP_065099.1 (H. sapiens) retinitispigmentosa GTPase regulator interacting protein 1; RPGR-interactingprotein [Homo sapiens] 512 1367930_at NM_017195 growth associatedprotein 43 513 1367628_at NM_019904 “lectin, galactose binding, soluble1” 514 1388112_at BG666999 solute carrier family 25 (mitochondrialadenine nucleotide translocator) member 4 515 1387873_at BI279661 wapfour-disulfide core domain 1 516 1389194_at AI406491 Rattus norvegicustranscribed sequences 517 1388928_at BF399310 Rattus norvegicustranscribed sequence with strong similarity to protein ref: NP_068733.1(H. sapiens) cofilin 2 (muscle) [Homo sapiens] 518 1370857_at BI282702smooth muscle alpha-actin 519 1387018_at NM_053770 Arg/Abl-interactingprotein ArgBP2 520 1398327_at BM386598 Rattus norvegicus transcribedsequence with strong similarity to protein pir: S69890 (H. sapiens)S69890 mitogen inducible gene mig-2 - human 521 1389189_at BF555956Rattus norvegicus transcribed sequence with weak similarity to proteinsp: Q9Z1P2 (R. norvegicus) AAC1_RAT Alpha-actinin 1 (Alpha-actinincytoskeletal isoform) (Non-muscle alpha-actinin 1) (F-actin crosslinking protein) 522 1371361_at BI278826 tensin 523 1375349_at BI295776Rattus norvegicus transcribed sequence with strong similarity to proteinpir: T17257 (H. sapiens) T17257 hypothetical protein DKFZp586P1422.1 -human 524 1376572_a_at AI045848 “Rattus norvegicus transcribed sequencewith moderate similarity to protein ref: NP_068506.1 (H. sapiens)supervillin, isoform 2; membrane-associated F-actin binding proteinp205; archvillin [Homo sapiens]” 525 1367813_at NM_130403 “proteinphosphatase 1, regulatory (inhibitor) subunit 14a” 526 1367648_atNM_013122 Insulin-like growth factor binding protein 2 527 1374969_atAA799832 Rattus norvegicus transcribed sequences 528 1371954_at BF290193Rattus norvegicus transcribed sequences 529 1370347_at AF095585 enigma(LIM domain protein) 530 1388483_at BI296011 Rattus norvegicustranscribed sequence with strong similarity to protein ref: NP_068733.1(H. sapiens) cofilin 2 (muscle) [Homo sapiens] 531 1375890_at BI296994Rattus norvegicus transcribed sequences 532 1368724_a_at NM_019131“tropomyosin 1, alpha” 533 1370288_a_at AF372216 “tropomyosin 1, alpha”534 1367785_at NM_031747 Calponin 1 535 1388422_at BI275904 Rattusnorvegicus transcribed sequence with moderate similarity to protein pir:JC2324 (H. sapiens) JC2324 LIM protein - human 536 1368988_at AW520914calsequestrin 2 537 1370585_a_at X04440 “protein kinase C, beta 1” 5381389187_at BI286421 Rattus norvegicus transcribed sequence with weaksimilarity to protein ref: NP_115950.1 (H. sapiens) EVG1 protein [Homosapiens] 539 1389050_at AI170797 Rattus norvegicus transcribed sequences540 1372658_at BG373779 desmuslin 541 1387898_at D29960 heat shock20-kDa protein 542 1373915_at AI044427 “Rattus norvegicus transcribedsequence with moderate similarity to protein pir: B49364 (H. sapiens)B49364 protein kinase (EC 2.7.1.37), myotonic dystrophy- associated -human” 543 1367570_at NM_031549 Transgelin (Smooth muscle 22 protein)544 1370287_a_at M23764 “tropomyosin 1, alpha” 545 1386869_at NM_012893“actin, gamma 2” 546 1388842_at BG380385 Rattus norvegicus transcribedsequences 547 1371382_at BI283060 “Rattus norvegicus transcribedsequence with strong similarity to protein pir: A37098 (H. sapiens)A37098 gelation factor ABP-280, long form - human” 548 1372219_atAA012755 “Rattus norvegicus transcribed sequence with strong similarityto protein sp: P06468 (H. sapiens) TPM2_HUMAN Tropomyosin beta chain,fibroblast and epithelial muscle-type (Tropomyosin 2, fibroblast andepithelial muscle-type) (TM36) (TME1) (TM1)” 549 1370057_at NM_017148cysteine rich protein 1 550 1371541_at AI177055 Rattus norvegicustranscribed sequence with moderate similarity to protein ref:NP_444278.1 (H. sapiens) mitochondrial ribosomal protein L53 [Homosapiens] 551 1388496_at AI103600 “Rattus norvegicus transcribed sequencewith strong similarity to protein ref: NP_001449.1 (H. sapiens) gammafilamin; filamin C, gamma (actin-binding protein- 280); filamin 2;actin-binding protein 280 [Homo sapiens]” 552 1370896_a_at X16262 myosinheavy chain 11 553 1372015_at AI008689 Rattus norvegicus transcribedsequence with weak similarity to protein sp: O75410 (H. sapiens)TAC1_HUMAN Transforming acidic coiled-coil- containing protein 1 5541372296_at AA800892 Rattus norvegicus transcribed sequence with weaksimilarity to protein sp: P55822 (H. sapiens) SH3B_HUMAN SH3domain-binding glutamic acid-rich protein (SH3BGR protein) (21-glutamicacid-rich protein) (21-GARP) 555 1388451_at AA817802 Rattus norvegicustranscribed sequences 556 1388298_at BI279044 “Rattus norvegicustranscribed sequence with strong similarity to protein pir: A32031 (H.sapiens) A32031 myosin regulatory light chain, smooth muscle - human”557 1371677_at BE113200 Rattus norvegicus transcribed sequences 5581367691_at NM_134449 PKC-delta binding protein 559 1372159_at BI285456Rattus norvegicus transcribed sequence with strong similarity to proteinref: NP_067541.1 (M. musculus) junctophilin 2 [Mus musculus] 5601371331_at BG665037 Rattus norvegicus transcribed sequences 5611370234_at AA893484 Fibronectin 1 562 1372111_at BI285449 caveolin 5631399065_at BM389543 Rattus norvegicus transcribed sequences 5641371855_at BM390254 Rattus norvegicus transcribed sequences 5651389394_at AI411809 Rattus norvegicus transcribed sequences 5661368145_at NM_013002 Neuron specific protein PEP-19 (Purkinje cellprotein 4) 567 1371566_at BI296437 Rattus norvegicus transcribedsequence with weak similarity to protein ref: NP_116264.1 (H. sapiens)hypothetical protein MGC15482 [Homo sapiens] 568 1368105_at AI228231Tspan-2 protein 569 1372625_at AA851663 Rattus norvegicus transcribedsequences 570 1372537_at BI289642 Rattus norvegicus transcribed sequencewith strong similarity to protein sp: O00423 (H. sapiens) EML1_HUMANEchinoderm microtubule-associated protein-like 1 (EMAP-1) (HuEMAP-1) 5711390430_at BF284190 “nuclear receptor subfamily 1, group D, member 2”572 1371969_at BI291848 Rattus norvegicus transcribed sequences 5731386860_at NM_012811 milk fat globule-EGF factor 8 protein 5741371588_at AA686007 “parvin, alpha” 575 1387224_at NM_019304“diacylglycerol kinase, beta” 576 1374237_at BI286025 “Rattus norvegicustranscribed sequence with strong similarity to protein sp: P29536 (H.sapiens) LMD1_HUMAN Leiomodin 1 (Leiomodin, muscle form) (64 kDaautoantigen D1) (64 kDa autoantigen 1D) (64 kDa autoantigen 1D3)(Thyroid-associated ophthalmopathy aut 577 1373911_at BM389026 Rattusnorvegicus transcribed sequence with moderate similarity to protein pir:S36110 (H. sapiens) S36110 osteoblast-specific factor 2 - human 5781371732_at BI285485 Rattus norvegicus transcribed sequences 5791371700_at AI177059 Rattus norvegicus transcribed sequence with strongsimilarity to protein sp: P55083 (H. sapiens) MFA4_HUMANMicrofibril-associated glycoprotein 4 580 1376099_at AW254017 “collagen,type V, alpha 1” 581 1388935_at AI231814 Rattus norvegicus transcribedsequences 582 1379936_at AA875132 Rattus norvegicus transcribedsequences 583 1372342_at AI176583 Rattus norvegicus transcribedsequences 584 1398370_at AW522471 rexo70 585 1369652_at AI145313 Thymuscell surface antigen 586 1376640_at BF285466 Rattus norvegicustranscribed sequences 587 1369955_at NM_134452 “collagen, type V, alpha1” 588 1373858_at BE109064 “Rattus norvegicus transcribed sequence withmoderate similarity to protein pdb: 1LBG (E. coli) B Chain B, LactoseOperon Repressor Bound To 21-Base Pair Symmetric Operator Dna, AlphaCarbons Only” 589 1387854_at BI282748 “procollagen, type I, alpha 2” 5901386862_at NM_013132 annexin 5 591 1388116_at BI285575 “collagen, type1, alpha 1” 592 1370959_at BI275716 “collagen, type III, alpha 1” 5931370155_at BM388837 “procollagen, type I, alpha 2” 594 1388569_atAI179984 alpha-2 antiplasmin 595 1373032_at AW251450 fracture callusprotein MUSTANG 596 1376265_at AI411542 Rattus norvegicus transcribedsequences 597 1389367_at AI409747 Rattus norvegicus transcribed sequencewith strong similarity to protein ref: NP_055390.1 (H. sapiens)schwannomin interacting protein 1 [Homo sapiens] 598 1373957_at BF281544Reelin 599 1370927_at BE108345 “procollagen, type XII, alpha 1” 6001372305_at AA893634 Rattus norvegicus transcribed sequence with moderatesimilarity to protein ref: NP_057513.1 (H. sapiens) COPZ2 fornonclathrin coat protein zeta-COP [Homo sapiens] 601 1383822_at AI029990“Rattus norvegicus transcribed sequence with weak similarity to proteinref: NP_001705.1 (H. sapiens) Bicaudal D homolog 1 (Drosophila);Bicaudal-D, Drosophila, homolog of, 1; Bicaudal D (Drosophila) homolog 1[Homo sapiens]” 602 1374774_at BF552241 Rattus norvegicus transcribedsequences 603 1388312_at BI274487 tissue inhibitor of metalloproteinase2 604 1376662_at BF390141 Rattus norvegicus transcribed sequences 6051374971_at AA818954 Rattus norvegicus transcribed sequences 6061389066_at BI274408 Rattus norvegicus transcribed sequences 6071373102_at BI282750 Rattus norvegicus transcribed sequences 6081388738_at AI411227 Rattus norvegicus transcribed sequences 6091368842_at BG377130 Rattus norvegicus transcribed sequences 6101390444_at AI044433 Rattus norvegicus transcribed sequences 6111377087_at AA963029 Rattus norvegicus transcribed sequences 6121387042_at NM_012828 “calcium channel, voltage-dependent, beta 3subunit” 613 1369974_at NM_012663 vesicle-associated membrane protein 2614 1374870_at BF286402 Rattus norvegicus transcribed sequences 6151374087_at AI411088 Rattus norvegicus transcribed sequences 6161374117_at BI279562 brain-specific angiogenesis inhibitor 1-associatedprotein 2 617 1390016_at BF415854 Rattus norvegicus transcribedsequences 618 1390341_at BF396709 Rattus norvegicus transcribedsequences 619 1374709_at AI406795 Rattus norvegicus transcribedsequences 620 1374726_at AI411941 Rattus norvegicus transcribedsequences 621 1389511_s_at BF403383 synaptogyrin 1 622 1390489_atBE108860 Rattus norvegicus transcribed sequences 623 1388598_at BI281230Rattus norvegicus transcribed sequences 624 1370375_at J05499 livermitochondrial glutaminase 625 1388821_at AI010430 Rattus norvegicustranscribed sequences 626 1368276_at NM_012664 Synaptophysin 6271375862_at BM384701 Rattus norvegicus transcribed sequence with weaksimilarity to protein sp: P07202 (H. sapiens) PERT_HUMAN Thyroidperoxidase precursor (TPO) 628 1377136_at AW254190 Rattus norvegicustranscribed sequences 629 1392887_at AI575082 Rattus norvegicustranscribed sequences 630 1389157_at BI275583 Rattus norvegicustranscribed sequences 631 1377013_at AI639108 Rattus norvegicustranscribed sequences 632 1371389_at AI170668 Rattus norvegicustranscribed sequences 633 1370969_at BE107303 Rattus norvegicustranscribed sequence with moderate similarity to protein sp: P03845 (E.coli) YPA1_ECOLI HYPOTHETICAL PROTEIN 1 634 1373438_at BE329352 Rattusnorvegicus transcribed sequence with weak similarity to protein pir:RGECDW (E. coli) RGECDW transcription activator of D-serinedehydratase - Escherichia coli 635 1388456_at AI228548 “Rattusnorvegicus transcribed sequence with strong similarity to protein sp:P23297 (H. sapiens) S10A_HUMAN S-100 protein, alpha chain (S100calcium-binding protein A1)” 636 1373427_at BI288816 Rattus norvegicustranscribed sequence with strong similarity to protein ref: NP_067067.1(H. sapiens) Rag D protein; hypothetical GTP-binding proteinDKFZp761H171 [Homo sapiens] 637 1368085_at NM_133595 GTP cyclohydrolaseI feedback regulatory protein 638 1369103_at NM_012755 Fynproto-oncogene 639 1389456_at BI296591 Rattus norvegicus transcribedsequences 640 1370904_at BI301490 R. norvegicus mRNA for RT1.Ma 6411371499_at AI227627 CD9 antigen (p24) 642 1371362_at BI285402 Rattusnorvegicus transcribed sequence with strong similarity to protein sp:Q92841 (H. sapiens) DD17_HUMAN Probable RNA-dependent helicase p72(DEAD-box protein p72) (DEAD-box protein 17) 643 1388920_at AI230985bone morphogenetic protein 6 644 1368851_at NM_012555 v-etserythroblastosis virus E26 oncogene homolog 1 (avian) 645 1373343_atAI175820 Rattus norvegicus transcribed sequences 646 1374888_at AI317837Rattus norvegicus transcribed sequences 647 1376453_at BF419074 Rattusnorvegicus transcribed sequences 648 1376062_at BG375315 syndecan 1 6491371046_at AW920849 “Rattus norvegicus beta II spectrin-short isoformmRNA, partial cds” 650 1368080_at NM_054008 Rgc32 protein 651 1371931_atBI274753 general transcription factor II I 652 1388463_at AW252660Rattus norvegicus transcribed sequence with moderate similarity toprotein ref: NP_057010.1 (H. sapiens) putative secreted protein; similarto putative secreted protein (H. sapiens) [Homo sapiens] 653 1388639_atBF284148 Rattus norvegicus transcribed sequence with strong similarityto protein ref: NP_060149.1 (H. sapiens) hypothetical protein FLJ20128[Homo sapiens] 654 1379685_at AI502753 CTD-binding SR-like protein rA4655 1389967_at AA892386 “Rattus norvegicus ADP-ribosylation-like factor6- interacting protein mRNA, complete cds” 656 1368941_at NM_032076prostaglandin E receptor 4 (subtype EP4) 657 1390457_at BM385762 Rattusnorvegicus transcribed sequences 658 1390458_at BG666849 Rattusnorvegicus transcribed sequences 659 1375254_at BE103444 Rattusnorvegicus transcribed sequence with strong similarity to protein pir:JC5023 (H. sapiens) JC5023 CMP-sialic acid transporter - human 6601375530_at BG374612 Rattus norvegicus transcribed sequence with strongsimilarity to protein ref: NP_062298.1 (M. musculus)glucosamine-phosphate N-acetyltransferase 1; glucosamine-6-phosphateacetyltransferase; glucosamine- phosphate N-acetyltransferase [Musmusculus]

TABLE 6 Genes differentially expressed in CNI1493-treated S1 DRG No.Identifier Acce. No. CTRL-VHL CTRL-CNI IBS-VHL IBS-CNI Description 521373932_at BE098739 1.00 4.46 1.91 6.75 ESTs 48 1370913_at AI409634 1.001.70 0.98 3.44 Best5 protein 15 1369202_at NM_017028 1.00 2.20 0.35 3.04myxovirus (influenza virus) resistance 2 62 1371152_a_at Z18877 1.001.49 0.48 2.48 25 oligoadenylate synthetase 53 1367850_at NM_053843 1.001.66 1.01 2.32 “Fc receptor, IgG, low affinity III” 54 1389006_atAI170394 1.00 1.53 1.03 2.28 ESTs 58 1383564_at BF411036 1.00 1.34 0.522.26 “ESTs, Highly similar to IRF7 MOUSE Interferon regulatory factor 7(IRF-7) [M. musculus]” 67 1389034_at BI295179 1.00 1.49 0.59 2.24 “ESTs,Moderately similar to UBPI_MOUSE Ubiquitin carboxyl-terminal hydrolase18 (Ubiquitin thiolesterase 18) (Ubiquitin-specific processing protease18) (Deubiquitinating enzyme 18) (43 kDa ubiquitin- specific protease)[M. musculus]” 35 1386166_at AA892881 1.00 1.59 1.43 2.18 EST 691388164_at AF029241 1.00 1.52 0.97 2.09 “Rattus norvegicus partial mRNAfor BM1k MHC class Ib antigen, strain SHR” 65 1370892_at BI285347 1.001.66 0.69 2.05 Complement component 4 73 1369186_at D85899 1.00 1.781.10 2.05 caspase 1 56 1372691_at BI292558 1.00 1.34 0.94 2.02 “ESTs,Highly similar to A57501 uridine phosphorylase (EC 2.4.2.3) I - mouse[M. musculus]” 70 1371015_at X52711 1.00 1.40 0.81 1.98 “Myxovirus(influenza) resistance, homolog of murine Mx (also interferon-inducibleprotein IFI78)” 74 1376151_a_at AI407953 1.00 1.51 0.92 1.95 ESTs 511374730_at AI102519 1.00 1.42 1.11 1.91 “ESTs, Highly similar to TYROprotein tyrosine kinase binding protein; killer cell activating receptorassociated protein [Mus musculus] [M. musculus]” 30 1390730_at BM3839111.00 1.69 1.33 1.86 ESTs 36 1375346_at BI290002 1.00 1.62 1.43 1.82“ESTs, Weakly similar to hypothetical protein FLJ20010 [Homo sapiens][H. sapiens]” 72 1390738_at BM385476 1.00 1.66 1.20 1.74 ESTs 471370186_at AI599350 1.00 1.32 1.05 1.74 “proteosome (prosome, macropain)subunit, beta type 9 (large multifunctional protease 2)” 80 1389553_atBF393825 1.00 1.16 0.85 1.73 “ESTs, Weakly similar to RIKEN cDNA1810046124 [Mus musculus] [M. musculus]” 77 1372930_at AI411381 1.001.14 0.76 1.72 “ESTs, Weakly similar to DEAF-1 related transcriptionalregulator (NUDR) [Rattus norvegicus] [R. norvegicus]” 59 1367786_atNM_080767 1.00 1.23 0.73 1.66 “proteasome (prosome, macropain) subunit,beta type, 8 (low molecular mass polypeptide 7)” 49 1388071_x_at M240241.00 1.28 1.05 1.66 RT1 class Ib gene 44 1380030_at AW523520 1.00 1.490.96 1.65 “ESTs, Highly similar to RIKEN cDNA 3110024A21 [Mus musculus][M. musculus]” 57 1390510_at BI294706 1.00 1.21 0.83 1.65 “ESTs, Weaklysimilar to RIKEN cDNA 1810027D10 [Mus musculus] [M. musculus]” 421374944_at BI275829 1.00 1.26 0.65 1.65 “ESTs, Moderately similar tohypothetical protein LOC51058 [Homo sapiens] [H. sapiens]” 23 1384446_atBF402271 1.00 1.72 1.51 1.62 ESTs 34 1375988_at AW914928 1.00 1.39 1.381.62 ESTs 27 1373403_at AI230625 1.00 1.59 1.11 1.61 ESTs 661387995_a_at BI285494 1.00 1.40 0.84 1.60 interferon-inducible proteinvariant 10 39 1387969_at U22520 1.00 1.52 1.06 1.59 “small induciblecytokine B subfamily (Cys-X-Cys), member 10” 32 1384167_at AW522260 1.001.70 1.38 1.59 ESTs 37 1387566_at NM_133551 1.00 1.42 1.25 1.59“phospholipase A2, group IVA (cytosolic, calcium- dependent)” 551372516_at AI317842 1.00 1.24 1.06 1.58 “ESTs, Weakly similar to S62328kinesin-like DNA binding protein KID - human [H. sapiens]” 43 1374277_atBI289615 1.00 1.44 1.01 1.56 ESTs 28 1376264_at AW526697 1.00 1.56 1.511.56 ESTs 33 1388872_at BI290053 1.00 1.25 1.19 1.56isopentenyl-diphosphate delta isomerase 14 1390312_at BG670441 1.00 1.450.77 1.55 “ESTs, Weakly similar to hypothetical protein FLJ20073 [Homosapiens] [H. sapiens]” 64 1388149_at X57523 1.00 1.15 0.72 1.52“Transporter 1, ABC (ATP binding cassette)” 71 1389571_at BG666368 1.001.20 0.95 1.52 “ESTs, Weakly similar to signal transducer and activatorof transcription 1 [Rattus norvegicus] [R. norvegicus]” 68 1368835_atAW434718 1.00 1.33 0.87 1.51 signal transducer and activator oftranscription 1 60 1388347_at AI233210 1.00 1.11 0.93 1.51 “ESTs,Moderately similar to I49013 thymic shared antigen- 1 - mouse [M.musculus]” 12 1376693_at AA998964 1.00 1.27 0.60 1.49 “ESTs, Moderatelysimilar to hypothetical protein FLJ20073 [Homo sapiens] [H. sapiens]” 501373043_at BI275923 1.00 1.36 1.16 1.49 “ESTs, Highly similar to stromalcell-derived factor 2- like 1 [Mus musculus] [M. musculus]” 241374465_at AI237098 1.00 1.48 1.04 1.49 “ESTs, Highly similar toubiquitously expressed transcript [Mus musculus] [M. musculus]” 631373025_at AI411618 1.00 1.15 0.61 1.47 “ESTs, Weakly similar to S49158complement protein C1q beta chain precursor - rat [R. norvegicus]” 251369962_at NM_031014 1.00 1.58 1.36 1.47 5-aminoimidazole-4- carboxamideribonucleotide formyltransferase/IMP 19 1377117_at BF387780 1.00 1.880.94 1.45 ESTs 16 1388754_at AI176839 1.00 1.49 0.88 1.45 ESTs 461376056_at BF291214 1.00 1.33 1.07 1.43 “ESTs, Moderately similar tohypothetical protein FLJ14464 [Homo sapiens] [H. sapiens]” 29 1399056_atAI716436 1.00 1.57 1.21 1.43 “ESTs, Moderately similar to candidatetumor suppressor protein [Homo sapiens] [H. sapiens]” 3 1376751_atAI044250 1.00 1.74 1.16 1.41 “ESTs, Highly similar to ST19_MOUSESerine/threonine-protein kinase 19 (RP1 protein) [M. musculus]” 111367846_at NM_012618 1.00 1.59 0.95 1.41 S100 calcium-binding protein A445 1373504_at BF287967 1.00 1.32 0.96 1.40 “ESTs, Weakly similar toJE0204 testicular protein Tpx- 1 - rat [R. norvegicus]” 22 1372043_atBI282363 1.00 1.52 1.16 1.37 “ESTs, Moderately similar to ribosomalprotein P0-like protein; 60S acidic ribosomal protein PO [Homo sapiens][H. sapiens]” 38 1370214_at AI175539 1.00 1.18 0.84 1.34 Parvalbumin(calcium binding protein) 9 1390655_at BF420653 1.00 1.47 0.54 1.34“ESTs, Highly similar to T14155 zinc finger protein Peg3 - mouse [M.musculus]” 26 1375669_at BI285619 1.00 1.39 1.03 1.32 “ESTs, Weaklysimilar to FK506 binding protein 2 (13 kDa) [Rattus norvegicus] [R.norvegicus]” 21 1398884_at BM384924 1.00 1.71 1.10 1.32 “ESTs, Highlysimilar to prefoldin 5; EIG-1; c-myc binding protein MM-1; DNA segment,Chr 15, ERATO Doi 697, expressed [Mus musculus] [M. musculus]” 11368465_at NM_012892 1.00 1.66 1.12 1.30 amiloride-sensitive cationchannel 1 20 1371381_at AI007981 1.00 1.46 0.99 1.29 “ESTs, Moderatelysimilar to UCRX_HUMAN Ubiquinol- cytochrome C reductase complex 7.2 kDaprotein (Cytochrome C1, nonheme 7 kDa protein) (Complex III subunit X)(7.2 kDa cytochrome c1-associated protein subunit) (HSPC119) [H.sapiens]” 4 1372815_at BG673028 1.00 1.70 1.02 1.29 “ESTs, Highlysimilar to MGN_HUMAN Mago nashi protein homolog [M. musculus]” 411388628_at BI284430 1.00 1.30 0.84 1.29 “ESTs, Weakly similar toPSD7_MOUSE 26S proteasome non-ATPase regulatory subunit 7 (26Sproteasome regulatory subunit S12) (Proteasome subunit p40) (Mov34protein) [M. musculus]” 61 1373037_at BI279216 1.00 1.07 0.74 1.26“ESTs, Weakly similar to ubiquitin-conjugating enzyme E2D 2 [Rattusnorvegicus] [R. norvegicus]” 2 1374950_at BF400611 1.00 1.52 0.89 1.23ESTs 10 1386425_at H33472 1.00 1.27 0.73 1.23 EST 31 1390065_at BE0966851.00 1.53 0.86 1.22 ESTs 17 1390389_at BM385936 1.00 1.21 0.87 1.20 ESTs7 1369970_at NM_031827 1.00 1.45 0.89 1.18 vesicle-associated membraneprotein 8 (endobrevin) 13 1390562_s_at BE102350 1.00 1.09 0.68 1.15 ESTs8 1390147_at AI549079 1.00 1.26 0.75 1.12 “ESTs, Highly similar toT17232 hypothetical protein DKFZp434I116.1 - human (fragment) [H.sapiens]” 95 1369157_at NM_017229 1.00 1.05 1.77 1.09 phosphodiesterase3B 78 1387242_at NM_019335 1.00 0.96 2.62 0.95 “Protein kinase,interferon- inducible double stranded RNA dependent” 79 1377225_atBF400628 1.00 0.76 3.47 0.85 ESTs 18 1372137_at BI279854 1.00 1.13 0.731.03 “ESTs, Highly similar to GC5L_MOUSE GCN5-LIKE PROTEIN 1 [M.musculus]” 75 1393280_at AA874924 1.00 1.18 0.28 1.09 “ESTs, Moderatelysimilar to lymphocyte antigen 86 [Mus musculus] [M. musculus]” 761372604_at BI289459 1.00 1.55 0.08 1.29 “ESTs, Weakly similar toapolipoprotein L, 3; TNF- inducible protein CG12-1 [Homo sapiens] [H.sapiens]” 102 1374802_at AI010721 1.00 0.90 1.72 0.96 “ESTs, Moderatelysimilar to hypothetical protein FLJ20424 [Homo sapiens] [H. sapiens]” 401371171_at M10094 1.00 0.76 6.16 0.89 RT1 class Ib gene 88 1371725_atBM392410 1.00 0.56 1.01 0.95 ESTs 5 1368316_at NM_019158 1.00 1.22 0.710.91 aquaporin 8 89 1375084_at BF419780 1.00 0.64 1.12 0.90 ESTs 851398272_at NM_022860 1.00 0.66 0.99 0.90 beta-4N-acetylgalactosaminyltransferase 81 1374721_at AI178647 1.00 0.74 1.150.89 ESTs 90 1374398_at AI178787 1.00 0.68 1.10 0.86 “ESTs, Highlysimilar to suppressor of Ty 6 homolog (S. cerevisiae) [Mus musculus] [M.musculus]” 91 1369999_a_at NM_053601 1.00 0.68 0.95 0.84 neuronatin 61387658_at U93849 1.00 1.61 0.43 0.83 Eukaryotic elongation factor 2kinase 129 1387349_at NM_013028 1.00 0.56 0.76 0.81 Short staturehomeobox 2 94 1390347_at AW535909 1.00 0.72 1.02 0.80 ESTs 1001376944_at AI407163 1.00 0.94 1.34 0.79 ESTs 96 1369052_at NM_1333231.00 0.90 1.29 0.79 zinc finger protein 111 84 1376410_at BI291814 1.000.66 0.93 0.76 ESTs 118 1389193_at BM388083 1.00 0.60 0.83 0.76 ESTs 991376537_at AW435010 1.00 0.81 1.06 0.75 ESTs 136 1374655_at BG3780951.00 0.57 0.90 0.75 ESTs 97 1375983_at AI234287 1.00 0.77 1.21 0.74 ESTs92 1392890_at BG663460 1.00 0.59 1.24 0.74 ESTs 135 1369061_at NM_0539061.00 0.25 0.81 0.74 glutathione reductase 101 1376610_a_at BE109163 1.000.77 1.60 0.74 “ESTs, Weakly similar to G02540 nucleobindin - human [H.sapiens]” 103 1377823_at AW531363 1.00 1.04 1.60 0.74 ESTs 82 1388116_atBI285575 1.00 0.56 1.13 0.73 “collagen, type 1, alpha 1” 121 1377070_atBE098910 1.00 0.59 0.72 0.73 “ESTs, Moderately similar to S3B2_HUMANSplicing factor 3B subunit 2 (Spliceosome associated protein 145) (SAP145) (SF3b150) (Pre-mRNA splicing factor SF3b 145 kDa subunit) [H.sapiens]” 131 1369958_at NM_022542 1.00 0.59 0.77 0.72 rhoB gene 1301376288_at AI408455 1.00 0.64 0.78 0.71 ESTs 132 1391078_at BM3918561.00 0.56 0.66 0.71 “ESTs, Moderately similar to A56284 differentiation-specific element binding protein - mouse [M. musculus]” 113 1374593_atAA799421 1.00 0.66 0.74 0.70 “ESTs, Highly similar to KPCE_RAT PROTEINKINASE C, EPSILON TYPE (NPKC-EPSILON) [R. norvegicus]” 112 1372158_atBI295768 1.00 0.66 0.79 0.70 LRP16 protein 83 1370969_at BE107303 1.000.58 1.05 0.69 homeo box A5 93 1370583_s_at AY082609 1.00 0.73 0.98 0.69P-glycoprotein/multidrug resistance 1 98 1370139_a_at AB051214 1.00 0.871.16 0.68 zzN/A 87 1389919_at BI296756 1.00 0.60 0.97 0.67 “ESTs, Weaklysimilar to actopaxin [Rattus norvegicus] [R. norvegicus]” 117 1368242_atNM_013186 1.00 0.60 0.72 0.66 “Potassium voltage gated channel,Shab-related subfamily, member 1” 106 1379747_at AA866443 1.00 0.83 0.970.66 ESTs 141 1390626_at BF398788 1.00 0.66 1.04 0.66 ESTs 1331369113_at NM_019282 1.00 0.59 0.87 0.65 “cysteine knot superfamily 1,BMP antagonist 1” 137 1376320_at BF393051 1.00 0.75 1.08 0.65 ESTs 861387588_at NM_138890 1.00 0.47 1.13 0.65 zzN/A 148 1399138_at BG6728051.00 0.71 0.87 0.65 ESTs 105 1398311_a_at AF313464 1.00 0.92 1.13 0.64kinase D-interacting substance of 220 kDa 115 1375538_at AI230737 1.000.66 0.82 0.63 ESTs 140 1376204_at AW531412 1.00 0.71 0.94 0.63 ESTs 1111390538_at BF414192 1.00 0.64 0.80 0.63 ESTs 151 1374909_at BI2966261.00 0.69 0.90 0.62 “ESTs, Weakly similar to Gasz [Rattus norvegicus][R. norvegicus]” 146 1368769_at NM_031760 1.00 0.82 1.01 0.62“ATP-binding cassette, subfamily B (MDR/TAP), member 11” 107 1387929_atAB020504 1.00 0.76 0.82 0.61 PMF32 protein 149 1376464_at BE102505 1.000.79 0.79 0.60 ESTs 110 1375705_at AI103622 1.00 0.60 0.73 0.59 Guaninenucleotide-binding protein beta 1 128 1374084_at BE119993 1.00 0.68 0.640.59 ESTs 123 1390722_at AW531272 1.00 0.60 0.70 0.59 ESTs 1221373008_x_at BF386649 1.00 0.44 0.57 0.58 reticulon 4 receptor 1391376939_at BI284907 1.00 0.67 0.85 0.58 ESTs 119 1369059_at NM_0537051.00 0.55 0.78 0.58 “transient receptor potential- related protein,ChaK” 126 1392500_at AA957990 1.00 0.59 0.67 0.58 ESTs 152 1369048_atNM_017289 1.00 0.71 0.64 0.56 “gamma-aminobutyric acid A receptor,delta” 124 1375508_at BE095963 1.00 0.50 0.61 0.56 “ESTs, Highly similarto KIF2_MOUSE KINESIN- LIKE PROTEIN KIF2 [M. musculus]” 142 1368429_atNM_133615 1.00 0.60 0.81 0.56 “TAF9-like RNA polymerase II, TATA boxbinding protein (TBP)-associated factor, 31 kD” 144 1373981_at BI2997201.00 0.62 0.60 0.55 ESTs 114 1369285_at NM_031082 1.00 0.74 0.76 0.55geranylgeranyltransferase type I (GGTase-I) 108 1372804_at AI175555 1.000.80 0.87 0.54 “ESTs, Highly similar to hypothetical brain proteinsimilar to X96994 BR-1 protein (Helix pomatia) [Mus musculus] [M.musculus]” 134 1386299_at AI639471 1.00 0.50 0.86 0.53 EST 1381377362_at BF390409 1.00 0.43 0.81 0.51 ESTs 125 1387131_at AF1930151.00 0.44 0.55 0.50 “serine (or cysteine) proteinase inhibitor, clade I(neuroserpin), member 1” 147 1373778_at BE349670 1.00 0.83 0.92 0.46ESTs 145 1377030_at BF390550 1.00 0.59 0.82 0.45 ESTs 143 1389436_atAI236615 1.00 0.75 0.77 0.45 ESTs 116 1386181_at AI639056 1.00 0.51 0.850.44 EST 150 1371042_at BG664160 1.00 0.63 0.70 0.42 mitogen-activatedprotein kinase kinase kinase kinase 3 109 1387453_at NM_024489 1.00 0.490.83 0.39 zinc finger protein RIN ZF 127 1390368_at AI716535 1.00 0.400.46 0.34 ESTs 104 1372640_at BI277758 1.00 0.85 1.80 0.33 ESTs 1201369054_at NM_133518 1.00 0.48 0.60 0.25 rabphilin 3A

The human homologs of some of the rat CVHGs are provided in Table 7.

TABLE 7 Human homologs of rat CVHGs Nucleotide Amino acid Gene Locus IDsequence sequence desmin 1674 SEQ ID NO: 1 SEQ ID NO: 34 PEP-19 5121 SEQID NO: 2 SEQ ID NO: 35 IGFBP2 3485 SEQ ID NO: 3 SEQ ID NO: 36 ADAMTS19510 SEQ ID NO: 4 SEQ ID NO: 37 ARGBP2 8470 SEQ ID NO: 5 SEQ ID NO: 38stathmin-like 2 11075 SEQ ID NO: 6 SEQ ID NO: 39 myxovirus resistance 24600 SEQ ID NO: 7 SEQ ID NO: 40 IRF7 3665 SEQ ID NO: 8 SEQ ID NO: 41GBP2 2634 SEQ ID NO: 9 SEQ ID NO: 42 SLC28a2 9153 SEQ ID NO: 10 SEQ IDNO: 43 BDNF 627 SEQ ID NO: 11 SEQ ID NO: 44 phosphodiesterase 3B 5140SEQ ID NO: 12 SEQ ID NO: 45 TREK2 54207 SEQ ID NO: 13 SEQ ID NO: 46 trkA4914 SEQ ID NO: 14 SEQ ID NO: 47 IL1R1 3554 SEQ ID NO: 15 SEQ ID NO: 48EEF2k 29904 SEQ ID NO: 16 SEQ ID NO: 49 actin, gamma 2 72 SEQ ID NO: 17SEQ ID NO: 50 myosin heavy chain 11 4629 SEQ ID NO: 18 SEQ ID NO: 51MRCL3 10627 SEQ ID NO: 19 SEQ ID NO: 52 MRLC2 103910 SEQ ID NO: 20 SEQID NO: 53 Rho family GTPase 1 27289 SEQ ID NO: 21 SEQ ID NO: 54 HSPB13315 SEQ ID NO: 22 SEQ ID NO: 55 RIPK4 54101 SEQ ID NO: 23 SEQ ID NO: 56type I protein 80316 SEQ ID NO: 24 SEQ ID NO: 57 phosphatase inhibitortransgelin 6876 SEQ ID NO: 25 SEQ ID NO: 58 beta 1 integrin 3688 SEQ IDNO: 26 SEQ ID NO: 59 Desmuslin 23336 SEQ ID NO: 27 SEQ ID NO: 60 C/EBPdelta 1052 SEQ ID NO: 28 SEQ ID NO: 61 FBXL22 283807 SEQ ID NO: 29 SEQID NO: 62 AF427491 SEQ ID NO: 30 SEQ ID NO: 63 cig5 91543 SEQ ID NO: 31SEQ ID NO: 64 SAMD9 54809 SEQ ID NO: 32 SEQ ID NO: 65 IFI27 3429 SEQ IDNO: 33 SEQ ID NO: 66

CVHGs and CVHG Products as Markers for CVH and CVH-Related Disorders

In general, Table 3 and Table 5 provide CVHGs that are differentiallyexpressed at in the CVH colon relative to controls. These genes may be acomponent in the disease mechanism and can be used as markers fordiagnosing and monitoring CVH and CVH-related disorders. The CVHGs ofTables 3 and 5, as well as the corresponding CVHG products (CVHPN andCVHPP) may become novel therapeutic targets for the treatment andprevention of CVH and CVH-related disorders. Furthermore, the CVHGproducts themselves may be used for the treatment of CVH.

Accordingly, the present invention pertains to the use of the CVHGslisted in Tables 3 and 5, the transcribed polynucleotides (CVHPNs), andthe encoded polypeptides (CVHPPs) as markers for CVH and CVH-relateddisorders. Moreover, the use of expression profiles of these genes canindicate the presence of or a risk of CVH and CVH-related disorders.These markers are further useful to correlate differences in levels ofexpression with a poor or favorable prognosis of CVH and CVH-relateddisorders. In particular, the present invention is directed to the useof CVHGs and panels of CVHGs set forth in Tables 3 and 5 or homologsthereof. For example, panels of the CVHGs can be conveniently arrayed onsolid supports, i.e., biochips, such as the GeneChip®, for use in kits.The CVHGs can be used to assess the efficacy of a treatment or therapyof CVH and CVH-related disorders, or as a target for a treatment ortherapeutic agent. The CVHGs can also be used to produce antibodiesspecific to CVHG products, and to construct gene therapy vectors thatinhibit the development of CVH and CVH-related disorders. Therefore,without limitation as to mechanism, the invention is based in part onthe principle that modulation of the expression of the CVHGs of theinvention may ameliorate CVH and CVH-related disorders when they areexpressed at levels similar or substantially similar to normal(non-diseased) tissue.

In one aspect, the invention provides CVHGs whose level of expression,which signifies their quantity or activity, is correlated with thepresence of CVH and CVH-related disorders. In certain preferredembodiments, the invention is performed by detecting the presence of anCVHPN or a CVHPP.

In another aspect of the invention, the expression levels of the CVHGsare determined in a particular subject sample for which either diagnosisor prognosis information is desired. The level of expression of a numberof CVHGs simultaneously provides an expression profile, which isessentially a “fingerprint” of the presence or activity of an CVHG orplurality of CVHGs that is unique to the state of the cell. In certainembodiments, comparison of relative levels of expression is indicativeof the severity of CVH and CVH-related disorders, and as such permitsfor diagnostic and prognostic analysis. Moreover, by comparing relativeexpression profiles of CVHGs from tissue samples taken at differentpoints in time, e.g., pre- and post-therapy and/or at different timepoints within a course of therapy, information regarding which genes areimportant in each of these stages is obtained. The identification ofgenes that are abnormally expressed in CVH versus normal tissue, as wellas differentially expressed genes during CVH development, allows the useof this invention in a number of ways. For example, comparison ofexpression profiles of CVHGs at different stages of the diseaseprogression provides a method for long-term prognosis. In anotherexample mentioned above, the efficacy of a particular treatment regimemay be evaluated, including whether a particular drug will act toimprove the long-term prognosis in a particular patient.

Similarly, CVHGs listed in Tables 4, 6 and 7 can also be used as targetsof CVH treatment and as markers to monitor the efficacy of CVHtreatment. The gene products from CVHGs of Tables 4, 6 and 7 may also beused in the treatment of CVH and CVH-related disorders.

The discovery of these differential expression patterns for individualor panels of CVHGs allows for screening test compounds with the goal ofmodulating a particular expression pattern. For example, screening canbe done for compounds that will convert an expression profile for a poorprognosis to one for a better prognosis. In certain embodiments, thismay be done by making biochips comprising sets of the significant CVHGs,which can then be used in these screens. These methods can also be doneon the protein level; that is, protein expression levels of the CVHGscan be evaluated for diagnostic and prognostic purposes or to screentest compounds. For example, in relation to these embodiments,significant CVHGs may comprise CVHGs which are determined to havemodulated activity or expression in response to a therapy regime.Alternatively, the modulation of the activity or expression of a CVHGmay be correlated with the diagnosis or prognosis of CVH and CVH-relateddisorders.

In addition, the CVHGs listed in Tables 3-8 can be administered for genetherapy purposes, including the administration of antisense nucleicacids and RNAi. The CVHG products (including CVHPPs and CVHPNs) andmodulator of CVHG products (such as anti-CVHPP antibodies) can also beadministered as therapeutic drugs.

For example, the CVHG desmin has significantly increased expression inCVH tissue samples, relative to control tissue samples. The presence ofincreased mRNA for this gene (or any other CVHGs set forth in Tables 3and 5), and increased levels of the protein products of this gene (orany other CVHGs set forth in Tables 3 and 5) serve as markers for CVH.Accordingly, amelioration of CVH can be achieved by modulatingup-regulated CVH markers, such as desmin, to normal levels.

In another embodiment of the invention, a product of CVHG, either in theform of a polynucleotide or a polypeptide, can be used as a therapeuticcompound of the invention. In yet other embodiments, a modulator of CVHGexpression or the activity of an CVHG product may be used as atherapeutic compound of the invention, or may be used in combinationwith one or more other therapeutic compositions of the invention.Formulation of such compounds into pharmaceutical compositions isdescribed in subsections below. Administration of such a therapeutic maysuppress bioactivity of CVHG product, and therefore may be used toameliorate CVH.

Sources of CVHG Products

The CVHG products (CVHPNs and CVHPPs) of the invention may be isolatedfrom any tissue or cell of a subject. It will be apparent to one skilledin the art that bodily fluids, such as blood or feces, may also serve assources from which the CVHG product of the invention may be assessed. Abiological sample of the invention is obtained as a blood sample, aurine or feces sample, a colon biopsy sample. A biological sample maycomprise biological components such as blood plasma, serum,erythrocytes, leukocytes, blood platelets, lymphocytes, macrophages,fibroblast cells, mast cells, fat cells, neuronal cells, epithelialcells and the like. The tissue samples containing one or more of theCVHG product themselves may be useful in the methods of the invention,and one skilled in the art will be cognizant of the methods by whichsuch samples may be conveniently obtained, stored and/or preserved.

Isolated Polynucleotides

One aspect of the invention pertains to isolated polynucleotides.Another aspect of the invention pertains to isolated polynucleotidefragments sufficient for use as hybridization probes to identify a CVHPNin a sample, as well as nucleotide fragments for use as PCRprobes/primers of the amplification or mutation of the nucleic acidmolecules which encode the CVHPP of the invention.

A CVHPN molecule of the present invention, e.g., a polynucleotidemolecule having the nucleotide sequence of one of the CVHGs listed inTables 3-8, or homologs thereof, or a portion thereof, can be isolatedusing standard molecular biology techniques and the sequence informationprovided herein, as well as sequence information known in the art. Usingall or a portion of the polynucleotide sequence of one of the CVHGslisted Tables 3-8 (or a homolog thereof) as a hybridization probe, aCVHG of the invention or a CVHPN of the invention can be isolated usingstandard hybridization and cloning techniques.

A CVHPN of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The polynucleotide so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to CVHG nucleotide sequencesof the invention can be prepared by standard synthetic techniques, e.g.,using an automated DNA synthesizer.

Alternatively, there are numerous amplification techniques for obtaininga full length coding sequence from a partial cDNA sequence. Within suchtechniques, amplification is generally performed via PCR. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers are preferably 22-30 nucleotides inlength, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

One such amplification technique is inverse PCR, which uses restrictionenzymes to generate a fragment in the known region of the gene. Thefragment is then circularized by intramolecular ligation and used as atemplate for PCR with divergent primers derived from the known region.Within an alternative approach, sequences adjacent to a partial sequencemay be retrieved by amplification with a primer to a linker sequence anda primer specific to a known region. The amplified sequences aretypically subjected to a second round of amplification with the samelinker primer and a second primer specific to the known region. Avariation on this procedure, which employs two primers that initiateextension in opposite directions from the known sequence, is describedin WO 96/38591.

Another such technique is known as “rapid amplification of cDNA ends” orRACE. This technique involves the use of an internal primer and anexternal primer, which hybridizes to a polyA region or vector sequence,to identify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:11-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60, 1991). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

In another preferred embodiment, an isolated polynucleotide molecule ofthe invention comprises a polynucleotide molecule which is a complementof the nucleotide sequence of a CVHG listed in Tables 3-8, or homologthereof, a CVHPN of the invention, or a portion of any of thesenucleotide sequences. A polynucleotide molecule which is complementaryto such a nucleotide sequence is one which is sufficiently complementaryto the nucleotide sequence such that it can hybridize to the nucleotidesequence, thereby forming a stable duplex.

The polynucleotide molecule of the invention, moreover, can compriseonly a portion of the polynucleotide sequence of a CVHG, for example, afragment which can be used as a probe or primer. The probe/primertypically comprises a substantially purified oligonucleotide. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 7 or 15,preferably about 25, more preferably about 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 400 or more consecutive nucleotidesof a CVHG or a CVHPN of the invention.

Probes based on the nucleotide sequence of anCVHG or anCVHPN of theinvention can be used to detect transcripts or genomic sequencescorresponding to the CVHG or CVHPN of the invention. In preferredembodiments, the probe comprises a label group attached thereto, e.g.,the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as a part of adiagnostic kit for identifying cells or tissue which misexpress (e.g.,over- or under-express) a CVHG, or which have greater or fewer copies ofan CVHG. For example, a level of a CVHG product in a sample of cellsfrom a subject may be determined, or the presence of mutations ordeletions of a CVHG of the invention may be assessed.

The invention further encompasses polynucleotide molecules that differfrom the polynucleotide sequences of the CVHGs listed in Tables 3-8 butencode the same proteins as those encoded by the genes shown in Tables3-8 due to degeneracy of the genetic code.

The invention also specifically encompasses homologs of the CVHGs listedin Tables 3-8 of other species. Gene homologs are well understood in theart and are available using databases or search engines such as thePubmed-Entrez database.

The invention also encompasses polynucleotide molecules which arestructurally different from the molecules described above (i.e., whichhave a slight altered sequence), but which have substantially the sameproperties as the molecules above (e.g., encoded amino acid sequences,or which are changed only in non-essential amino acid residues). Suchmolecules include allelic variants, and are described in greater detailin subsections herein.

In addition to the nucleotide sequences of the CVHGs listed in Tables3-8, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof the proteins encoded by the CVHGs listed in Tables 3-8 may existwithin a population (e.g., the human population). Such geneticpolymorphism in the CVHGs listed in Tables 3-8 may exist amongindividuals within a population due to natural allelic variation. Anallele is one of a group of genes which occur alternatively at a givengenetic locus. In addition it will be appreciated that DNA polymorphismsthat affect RNA expression levels can also exist that may affect theoverall expression level of that gene (e.g., by affecting regulation ordegradation). As used herein, the phrase “allelic variant” includes anucleotide sequence which occurs at a given locus or to a polypeptideencoded by the nucleotide sequence.

Polynucleotide molecules corresponding to natural allelic variants andhomologs of the CVHGs can be isolated based on their homology to theCVHGs listed in Tables 3-8, using the cDNAs disclosed herein, or aportion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions.Polynucleotide molecules corresponding to natural allelic variants andhomologs of the CVHGs of the invention can further be isolated bymapping to the same chromosome or locus as the CVHGs of the invention.

In another embodiment, an isolated polynucleotide molecule of theinvention is at least 15, 20, 25, 30, 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more nucleotidesin length and hybridizes under stringent conditions to a polynucleotidemolecule corresponding to a nucleotide sequence of an CVHG of theinvention. Preferably, the isolated polynucleotide molecule of theinvention hybridizes under stringent conditions to the sequence of oneof the CVHGs set forth in Tables 3-8, or corresponds to anaturally-occurring polynucleotide molecule.

In addition to naturally-occurring allelic variants of the CVHG of theinvention that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of the CVHGs of the invention, thereby leading tochanges in the amino acid sequence of the encoded proteins, withoutaltering the functional activity of these proteins. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made. A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequence of a protein without altering the biological activity, whereasan “essential” amino acid residue is required for biological activity.For example, amino acid residues that are conserved among allelicvariants or homologs of a gene (e.g., among homologs of a gene fromdifferent species) are predicted to be particularly unamenable toalteration.

In yet other aspects of the invention, polynucleotides of a CVHG maycomprise one or more mutations. An isolated polynucleotide moleculeencoding a protein with a mutation in a CVHPP of the invention can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of the gene encoding theCVHPP, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Such techniques arewell known in the art. Mutations can be introduced into the CVHG of theinvention by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. Alternatively, mutations can be introduced randomly along allor part of a coding sequence of a CVHG of the invention, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

A polynucleotide may be further modified to increase stability in vivo.Possible modifications include, but are not limited to, the addition offlanking sequences at the 5′ and/or 3′ ends; the use of phosphorothioateor 2 O-methyl rather than phosphodiesterase linkages in the backbone;and/or the inclusion of nontraditional bases such as inosine, queosineand wybutosine, as well as acetyl- methyl-, thio- and other modifiedforms of adenine, cytidine, guanine, thymine and uridine.

Another aspect of the invention pertains to isolated polynucleotidemolecules, which are antisense to the CVHGs of the invention. An“antisense” polynucleotide comprises a nucleotide sequence which iscomplementary to a “sense” polynucleotide encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisensepolynucleotide can hydrogen bond to a sense polynucleotide. Theantisense polynucleotide can be complementary to an entire coding strandof a gene of the invention or to only a portion thereof. In oneembodiment, an antisense polynucleotide molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence of theinvention. The term “coding region” includes the region of thenucleotide sequence comprising codons which are translated into aminoacids. In another embodiment, the antisense polynucleotide molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence of the invention.

Antisense polynucleotides of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense polynucleotidemolecule can be complementary to the entire coding region of an mRNAcorresponding to a gene of the invention, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region. An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense polynucleotide of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense polynucleotide can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense polynucleotides, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense polynucleotide include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenosine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisensepolynucleotide can be produced biologically using an expression vectorinto which a polynucleotide has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted polynucleotide willbe of an antisense orientation to a target polynucleotide of interest,described further in the following subsection).

The antisense polynucleotide molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a CVHPP of theinvention to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex or, forexample, in the cases of an antisense polynucleotide molecule whichbinds to DNA duplexes, through specific interactions in the major grooveof the double helix. An example of a route of administration ofantisense polynucleotide molecules of the invention include directinjection at a tissue site. Alternatively, antisense polynucleotidemolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking theantisense polynucleotide molecules to peptides or antibodies which bindto cell surface receptors or antigens. The antisense polynucleotidemolecules can also be delivered to cells using the vectors describedherein. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs comprising the antisensepolynucleotide molecules are preferably placed under the control of astrong promoter.

In yet another embodiment, the antisense polynucleotide molecule of theinvention is an α-anomeric polynucleotide molecule. An α-anomericpolynucleotide molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other. The antisense polynucleotide molecule canalso comprise a 2′-o-methylribonucleotide or a chimeric RNA-DNAanalogue.

In still another embodiment, an antisense polynucleotide of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandedpolynucleotide, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes) can be used tocatalytically cleave mRNA transcripts of the CVHGs of the invention tothereby inhibit translation of the mRNA. A ribozyme having specificityfor a CVHPN can be designed based upon the nucleotide sequence of theCVHPN. For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in a CVHG mRNA.Alternatively, mRNA transcribed from a CVHG can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. Alternatively, expression of a CVHG of the invention can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the CVHG (e.g., the promoter and/or enhancers) toform triple helical structures that prevent transcription of the gene intarget cells.

Expression of the CVHGs of the invention can also be inhibited using RNAinterference (“RNA_(i)”). This is a technique for post-transcriptionalgene silencing (“PTGS”), in which target gene activity is specificallyabolished with cognate double-stranded RNA (“dsRNA”). RNA_(i) resemblesin many aspects PTGS in plants and has been detected in manyinvertebrates including the trypanosome, hydra, planaria, nematode andfruit fly (Drosophila melanogaster). It may be involved in themodulation of transposable element mobilization and antiviral stateformation. RNA_(i) technology is disclosed, for example, in U.S. Pat.No. 5,919,619 and PCT Publication Nos. WO99/14346 and WO01/29058.Basically, dsRNA of about 21 nucleotides, homologous to the target gene,is introduced into the cell and a sequence specific reduction in geneactivity is observed.

In yet another embodiment, the polynucleotide molecules of the presentinvention can be modified at the base moiety, sugar moiety or phosphatebackbone to improve the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of thepolynucleotide molecules can be modified to generate peptidepolynucleotides. As used herein, the terms “peptide polynucleotides” or“PNAs” refer to polynucleotide mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense agents for sequence-specificmodulation of CVHG expression by, for example, inducing transcription ortranslation arrest or inhibiting replication. PNAs of the polynucleotidemolecules of the invention can be used in the analysis of single basepair mutations in a gene, (e.g., by PNA-directed PCR clamping). They mayalso serve as artificial restriction enzymes when used in combinationwith other enzymes (e.g., S1 nucleases) or as probes or primers for DNAsequencing or hybridization.

In another embodiment, PNAs can be modified, (e.g., to enhance theirstability or cellular uptake), by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras of the polynucleotide molecules of theinvention can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation.The synthesis of PNA-DNA chimeras can be performed. For example, a DNAchain can be synthesized on a solid support using standardphosphoramidite coupling chemistry. Modified nucleoside analogs, such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a spacer between the PNA and the 5′ end of DNA. PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment. Alternatively, chimeric moleculescan be synthesized with a 5′ DNA segment and a 3′ PNA segment.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane or theblood-kidney barrier (see, e.g. PCT Publication No. W089/10134). Inaddition, oligonucleotides can be modified with hybridization-triggeredcleavage agents or intercalating agents. To this end, theoligonucleotide may be conjugated to another molecule (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent). Finally, the oligonucleotidemay be detectably labeled, either such that the label is detected by theaddition of another reagent (e.g., a substrate for an enzymatic label),or is detectable immediately upon hybridization of the nucleotide (e.g.,a radioactive label or a fluorescent label).

Isolated Polypeptides

Several aspects of the invention pertain to isolated CVHPPs, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-CVHPP antibodies. In oneembodiment, native CVHPPs can be isolated from cells or tissue sourcesby an appropriate purification scheme using standard proteinpurification techniques. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example, aCVHPP may be purified using a standard anti-CVHPP antibody column.Ultrafiltration and diafiltration techniques, in conjunction withprotein concentration, are also useful. The degree of purificationnecessary will vary depending on the use of the CVHPP. In some instancesno purification will be necessary.

In another embodiment, CVHPPs or mutated CVHPPs are produced byrecombinant DNA techniques. Alternative to recombinant expression, aCVHPP or mutated CVHPP can be synthesized chemically using standardpeptide synthesis techniques.

The invention also provides variants of CVHPPs. The variant of a CVHPPis substantially homologous to the native CVHPP encoded by an CVHGlisted in Table 4, and retains the functional activity of the nativeCVHPP, yet differs in amino acid sequence due to natural allelicvariation or mutagenesis, as described in detail above. Accordingly, inanother embodiment, the variant of a CVHPP is a protein which comprisesan amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or more homologous to the amino acid sequence of the originalCVHPP.

In a non-limiting example, as used herein, proteins are referred to as“homologs” and “homologous” where a first protein region and a secondprotein region are compared in terms of identity. To determine thepercent identity of two amino acid sequences or of two polynucleotidesequences, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or polynucleotide sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleotide “identity” is equivalent to amino acidor nucleotide “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. 48:444-453, 1970) algorithm which has been incorporated intothe GAP program in the GCG software package, using either a Blossom 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package, using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6.

The polynucleotide and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using BLAST programs availableat the BLAST website maintained by the National Center of biotechnologyInformation (NCBI), National Library of Medicine, Washington D.C. USA.

The invention also provides chimeric or fusion CVHPPs. Within a fusionCVHPP the polypeptide can correspond to all or a portion of a CVHPP. Ina preferred embodiment, a fusion CVHPP comprises at least onebiologically active portion of a CVHPP. Within the fusion protein, theterm “operatively linked” is intended to indicate that the CVHPP-relatedpolypeptide and the non-CVHPP-related polypeptide are fused in-frame toeach other. The non-CVHPP-related polypeptide can be fused to theN-terminus or C-terminus of the CVHPP-related polypeptide.

A peptide linker sequence may be employed to separate the CVHPP-relatedpolypeptide from non-CVHPP-related polypeptide components by a distancesufficient to ensure that each polypeptide folds into its secondary andtertiary structures. Such a peptide linker sequence is incorporated intothe fusion protein using standard techniques well known in the art.Suitable peptide linker sequences may be chosen based on the followingfactors: (1) their ability to adopt a flexible extended conformation;(2) their inability to adopt a secondary structure that could interactwith functional epitopes on the CVHPP-related polypeptide andnon-CVHPP-related polypeptide; and (3) the lack of hydrophobic orcharged residues that might react with the polypeptide functionalepitopes. Preferred peptide linker sequences contain gly, asn and serresidues. Other near neutral amino acids, such as thr and ala may alsobe used in the linker sequence. Amino acid sequences which may be usedas linkers are well known in the art. The linker sequence may generallybe from 1 to about 50 amino acids in length. Linker sequences are notrequired when the CVHPP-related polypeptide and non-CVHPP-relatedpolypeptide have non-essential N-terminal amino acid regions that can beused to separate the functional domains and prevent steric interference.

For example, in one embodiment, the fusion protein is a glutathioneS-transferase (GST)-CVHPP fusion protein in which the CVHPP sequencesare fused to the C-terminus of the GST sequences. Such fusion proteinscan facilitate the purification of recombinant CVHPPs.

The CVHPP-fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo, asdescribed herein. The CVHPP-fusion proteins can be used to affect thebioavailability of a CVHPP substrate. CVHPP-fusion proteins may beuseful therapeutically for the treatment of, or prevention of, damagescaused by, for example, (i) aberrant modification or mutation of a CVHG;(ii) mis-regulation of a CVHG; and (iii) aberrant post-translationalmodification of a CVHPP.

Moreover, the CVHPP-fusion proteins of the invention can be used asimmunogens to produce anti-CVHPP antibodies in a subject, to purifyCVHPP ligands, and to identify molecules which inhibit the interactionof a CVHPP with a CVHPP substrate in screening assays.

Preferably, a CVHPP-chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques. In anotherembodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence. Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A CVHPP-encoding polynucleotide can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theCVHPP.

A signal sequence can be used to facilitate secretion and isolation ofthe secreted protein or other proteins of interest. Signal sequences aretypically characterized by a core of hydrophobic amino acids which aregenerally cleaved from the mature protein during secretion in one ormore cleavage events. Such signal peptides contain processing sites thatallow cleavage of the signal sequence from the mature proteins as theypass through the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as topolypeptides from which the signal sequence has been proteolyticallycleaved (i.e., the cleavage products). In one embodiment, apolynucleotide sequence encoding a signal sequence can be operablylinked in an expression vector to a protein of interest, such as aprotein which is ordinarily not secreted or is otherwise difficult toisolate. The signal sequence directs secretion of the protein, such asfrom a eukaryotic host into which the expression vector is transformed,and the signal sequence is subsequently or concurrently cleaved. Theprotein can then be readily purified from the extracellular medium byart recognized methods. Alternatively, the signal sequence can be linkedto the protein of interest using a sequence which facilitatespurification, such as with a GST domain.

The present invention also pertains to variants of the CVHPPs of theinvention which function as either agonists or as antagonists to theCVHPPs. In one embodiment, antagonists or agonists of CVHPPs are used astherapeutic agents. For example, antagonists of an up-regulated CVHGthat can decrease the activity or expression of such a gene andtherefore ameliorate CVH in a subject wherein the CVHG is abnormallyincreased in level or activity. In this embodiment, treatment of such asubject may comprise administering an antagonist wherein the antagonistprovides decreased activity or expression of the targeted CVHG.

In certain embodiments, an agonist of the CVHPPs can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of a CVHPP or may enhance an activity of aCVHPP. In certain embodiments, an antagonist of a CVHPP can inhibit oneor more of the activities of the naturally occurring form of the CVHPPby, for example, competitively modulating an activity of a CVHPP. Thus,specific biological effects can be elicited by treatment with a variantof limited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring forth of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the CVHPP.

Mutants of a CVHPP which function as either CVHPP agonists or as CVHPPantagonists can be identified by screening combinatorial libraries ofmutants, e.g., truncation mutants, of a CVHPP for CVHPP agonist orantagonist activity. In certain embodiments, such mutants may be used,for example, as a therapeutic protein of the invention. A diverselibrary of CVHPP mutants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential CVHPP sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of CVHPP sequencestherein. There are a variety of methods which can be used to producelibraries of potential CVHPP variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene isthen ligated into an appropriate expression vector. Use of a degenerateset of genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential CVHPP sequences. Methodsfor synthesizing degenerate oligonucleotides are known in the art.

In addition, libraries of fragments of a protein coding sequencecorresponding to a CVHPP of the invention can be used to generate adiverse or heterogenous population of CVHPP fragments for screening andsubsequent selection of variants of a CVHPP. In one embodiment, alibrary of coding sequence fragments can be generated by treating adouble-stranded PCR fragment of a CVHPP coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule,denaturing the double-stranded DNA, renaturing the DNA to formdouble-stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single-stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the CVHPP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high-throughputanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify CVHPPvariants (Delgrave et al. Protein Engineering 6:327-331, 1993).

Portions of a CVHPP or variants of a CVHPP having less than about 100amino acids, and generally less than about 50 amino acids, may also begenerated by synthetic means, using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain.Equipment for automated synthesis of polypeptides is commerciallyavailable from suppliers such as Perkin Elmer/Applied BioSystemsDivision (Foster City, Calif.), and may be operated according to themanufacturer's instructions.

Methods and compositions for screening for protein inhibitors oractivators are known in the art (see U.S. Pat. Nos. 4,980,281,5,266,464, 5,688,635, and 5,877,007, which are incorporated herein byreference).

It is contemplated in the present invention that CVHPPs are cleaved intofragments for use in further structural or functional analysis, or inthe generation of reagents such as CVHPP and CVHPP-specific antibodies.This can be accomplished by treating purified or unpurified polypeptidewith a proteolytic enzyme (i.e., a proteinase) including, but notlimited to, serine proteinases (e.g., chymotrypsin, trypsin, plasmin,elastase, thrombin, substilin) metal proteinases (e.g., carboxypeptidaseA, carboxypeptidase B, leucine aminopeptidase, thermolysin,collagenase), thiol proteinases (e.g., papain, bromelain, Streptococcalproteinase, clostripain) and/or acid proteinases (e.g., pepsin,gastricsin, trypsinogen). Polypeptide fragments are also generated usingchemical means such as treatment of the polypeptide with cyanogenbromide (CNBr), 2-nitro-5-thiocyanobenzoic acid, isobenzoic acid,BNPA-skatole, hydroxylamine or a dilute acid solution. Recombinanttechniques are also used to produce specific fragments of a CVHPP.

In addition, the invention also contemplates that compounds stericallysimilar to a particular CVHPP may be formulated to mimic the keyportions of the peptide structure, called peptidomimetics or peptidemimetics. Mimetics are peptide-containing molecules which mimic elementsof polypeptide secondary structure. See, for example, U.S. Pat. No.5,817,879 (incorporated by reference hereinafter in its entirety). Theunderlying rationale behind the use of peptide mimetics is that thepeptide backbone of polypeptides exists chiefly to orient amino acidside chains in such a way as to facilitate molecular interactions, suchas those of receptor and ligand. Recently, peptide and glycoproteinmimetic antigens have been described which elicit protective antibody toNeisseria meningitidis serogroup B, thereby demonstrating the utility ofmimetic applications (Moe et al., Int. Rev. Immunol. 20:201-20, 2001;Berezin et al., J Mol Neurosci. 22:33-39, 2004). Successful applicationsof the peptide mimetic concept have thus far focused on mimetics ofβ-turns within polypeptides. Likely β-turn structures within a CVHPP canbe predicted by computer-based algorithms. For example, U.S. Pat. No.5,933,819, incorporated by reference hereinafter in its entirety,describes a neural network based method and system for identifyingrelative peptide binding motifs from limited experimental data. Inparticular, an artificial neural network (ANN) is trained with peptideswith known sequence and function (i.e., binding strength) identifiedfrom a phage display library. The ANN is then challenged with unknownpeptides, and predicts relative binding motifs. Analysis of the unknownpeptides validate the predictive capability of the ANN. Once thecomponent amino acids of the turn are determined, mimetics can beconstructed to achieve a similar spatial orientation of the essentialelements of the amino acid side chains, as discussed in U.S. Pat. No.6,420,119 and U.S. Pat. No. 5,817,879, and in Kyte and Doolittle, J.Mol. Biol., 157:105-132, 1982; Moe and Granoff, Int. Rev. Immunol.,20(2):201-20, 2001; Granoff et al., J. Immunol., 167(11):6487-96, 2001(each incorporated by reference hereinafter in its entirety).

Antibodies

In another aspect, the invention includes antibodies that are specificto CVHPPs of the invention or their variants. Preferably the antibodiesare monoclonal, and most preferably, the antibodies are humanized, asper the description of antibodies described below.

An isolated CVHPP, or a portion or fragment thereof, can be used as animmunogen to generate antibodies that bind the CVHPP using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length CVHPP can be used or, alternatively, the invention providesantigenic peptide fragments of the CVHPP for use as immunogens. Theantigenic peptide of a CVHPP comprises at least 8 amino acid residues ofan amino acid sequence encoded by an CVHG set forth in Tables 3-8 or anhomolog thereof, and encompasses an epitope of a CVHPP such that anantibody raised against the peptide forms a specific immune complex withthe CVHPP. Preferably, the antigenic peptide comprises at least 8 aminoacid residues, more preferably at least 12 amino acid residues, evenmore preferably at least 16 amino acid residues, and most preferably atleast 20 amino acid residues.

Immunogenic portions (epitopes) may generally be identified using wellknown techniques. Such techniques include screening polypeptides for theability to react with antigen-specific antibodies, antisera and/orT-cell lines or clones. As used herein, antisera and antibodies are“antigen-specific” if they bind to an antigen with a binding affinityequal to, or greater than 10⁵ M⁻¹. Such antisera and antibodies may beprepared as described herein, and using well known techniques. Anepitope of a CVHPP is a portion that reacts with such antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Such epitopes may react within such assays at a level that issimilar to or greater than the reactivity of the full lengthpolypeptide. Such screens may generally be performed using methods wellknown to those of ordinary skill in the art. For example, a polypeptidemay be immobilized on a solid support and contacted with patient sera toallow binding of antibodies within the sera to the immobilizedpolypeptide. Unbound sera may then be removed and bound antibodiesdetected using, for example, ¹²⁵I-labeled Protein A.

Preferred epitopes encompassed by the antigenic peptide are regions ofthe CVHPP that are located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity.

A CVHPP immunogen typically is used to prepare antibodies by immunizinga suitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed CVHPP or a chemically synthesizedCVHPP. The preparation can further include an adjuvant, such as Freund'scomplete or incomplete adjuvant, or a similar immunostimulatory agent.Immunization of a suitable subject with an immunogenic CVHPP preparationinduces a polyclonal anti-CVHPP antibody response. Techniques forpreparing, isolating and using antibodies are well known in the art.

Accordingly, another aspect of the invention pertains to monoclonal orpolyclonal anti-CVHPP antibodies and immunologically active portions ofthe antibody molecules, including F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin.

Polyclonal anti-CVHPP antibodies can be prepared as described above byimmunizing a suitable subject with a CVHPP. The anti-CVHPP antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized CVHPP. If desired, the antibody molecules directedagainst CVHPPs can be isolated from the subject (e.g., from the blood)and further purified by well known techniques, such as protein Achromatography, to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-CVHPP antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique, human B cell hybridoma technique, the EBV-hybridomatechnique, or trioma techniques. The technology for producing monoclonalantibody hybridomas is well known. Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a CVHPP immunogen as described above, andthe culture supernatants of the resulting hybridoma cells are screenedto identify a hybridoma producing a monoclonal antibody that binds to aCVHPP of the invention.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-CVHPP monoclonal antibody. Moreover, the ordinarily skilled workerwill appreciate that there are many variations of such methods whichalso would be useful. Typically, the immortal cell line (e.g., a myelomacell line) is derived from the same mammalian species as thelymphocytes. For example, murine hybridomas can be made by fusinglymphocytes from a mouse immunized with an immunogenic preparation ofthe present invention with an immortalized mouse cell line. Preferredimmortal cell lines are mouse myeloma cell lines that are sensitive toculture medium containing hypoxanthine, aminopterin and thymidine (“HATmedium”). Any of a number of myeloma cell lines can be used as a fusionpartner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,P3-x63-Ag8.653 or Sp210-Ag14 myeloma lines. These myeloma lines areavailable from ATCC. Typically, HAT-sensitive mouse myeloma cells arefused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridomacells resulting from the fusion are then selected using HAT medium,which kills unfused and unproductively fused myeloma cells (unfusedsplenocytes die after several days because they are not transformed).Hybridoma cells producing a monoclonal antibody of the invention aredetected by screening the hybridoma culture supernatants for antibodiesthat bind to a CVHPP, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-CVHPP antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phase display library) with CVHPP to thereby isolateimmunoglobulin library members that bind to a CVHPP. Kits for generatingand screening phage display libraries are commercially available.

The anti-CVHPP antibodies also include “Single-chain Fv” or “scFv”antibody fragments. The scFv fragments comprise the V_(H) and V_(L)domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding.

Additionally, recombinant anti-CVHPP antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art.

Humanized antibodies are particularly desirable for therapeutictreatment of human subjects. Humanized forms of non-human (e.g., murine)antibodies are chimeric molecules of immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies), which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesforming a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theconstant regions being those of a human immunoglobulin consensussequence. The humanized antibody will preferably also comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin.

Such humanized antibodies can be produced using transgenic mice whichare incapable of expressing endogenous immunoglobulin heavy and lightchain genes, but which can express human heavy and light chain genes.The transgenic mice are immunized in the normal fashion with a selectedantigen, e.g., all or a portion of a polypeptide corresponding to aCVHPP of the invention. Monoclonal antibodies directed against theantigen can be obtained using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA and IgE antibodies.

Humanized antibodies which recognize a selected epitope can be generatedusing a technique referred to as “guided selection.” In this approach aselected non-human monoclonal antibody, e.g., a murine antibody, is usedto guide the selection of a humanized antibody recognizing the sameepitope.

In a preferred embodiment, the antibodies to CVHPP are capable ofreducing or eliminating the biological function of CVHPP, as isdescribed below. That is, the addition of anti-CVHPP antibodies (eitherpolyclonal or preferably monoclonal) to CVHPP (or cells containingCVHPP) may reduce or eliminate the CVHPP activity. Generally, at least a25% decrease in activity is preferred, with at least about 50% beingparticularly preferred and about a 95-100% decrease being especiallypreferred.

An anti-CVHPP antibody can be used to isolate a CVHPP of the inventionby standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-CVHPP antibody can facilitate thepurification of natural CVHPPs from cells and of recombinantly producedCVHPPs expressed in host cells. Moreover, an anti-CVHPP antibody can beused to detect a CVHPP (e.g., in a cellular lysate or cell supernatanton the cell surface) in order to evaluate the abundance and pattern ofexpression of the CVHPP. Anti-CVHPP antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, for example, to determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialsinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Anti-CVHPP antibodies of the invention are also useful for targeting atherapeutic to a cell or tissue comprising the antigen of the anti-CVHPPantibody. For example, a therapeutic, such as a small molecule, can belinked to the anti-CVHPP antibody in order to target the therapeutic tothe cell or tissue comprising the CVHPP antigen. The method isparticularly useful in connection with CVHPPs which are surface markers.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional, may be employed as the linker group. Coupling may beeffected, for example, through amino groups, carboxyl groups, sulfhydrylgroups or oxidized carbohydrate residues. There are numerous referencesdescribing such methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwellet al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule. Alternatively, linkers that provide multiple sitesfor attachment can be used.

As is well known in the art, a given polypeptide or polynucleotide mayvary in its immunogenicity. It is often necessary therefore to couplethe immunogen (e.g., a polypeptide or polynucleotide) of the presentinvention with a carrier. Exemplary and preferred carriers are CRM197, Ecoli (LT) toxin, V. cholera (CT) toxin, keyhole limpet hemocyanin (KLH)and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouseserum albumin or rabbit serum albumin can also be used as carriers.

Where a CVHPP (or a fragment thereof) and a carrier protein areconjugated (i.e., covalently associated), conjugation may be anychemical method, process or genetic technique commonly used in the art.For example, a CVHPP (or a fragment thereof) and a carrier protein, maybe conjugated by techniques, including, but not limited to: (1) directcoupling via protein functional groups (e.g., thiol-thiol linkage,amine-carboxyl linkage, amine-aldehyde linkage; enzyme direct coupling);(2) homobifunctional coupling of amines (e.g., using bis-aldehydes); (3)homobifunctional coupling of thiols (e.g., using bis-maleimides); (4)homobifunctional coupling via photoactivated reagents (5)heterobifunctional coupling of amines to thiols (e.g., usingmaleimides); (6) heterobifunctional coupling via photoactivated reagents(e.g., the β-carbonyldiazo family); (7) introducing amine-reactivegroups into a poly- or oligosaccharide via cyanogen bromide activationor carboxymethylation; (8) introducing thiol-reactive groups into apoly- or oligosaccharide via a heterobifunctional compound such asmaleimido-hydrazide; (9) protein-lipid conjugation via introducing ahydrophobic group into the protein and (10) protein-lipid conjugationvia incorporating a reactive group into the lipid. Also, contemplatedare heterobifunctional “non-covalent coupling” techniques such theBiotin-Avidin interaction. For a comprehensive review of conjugationtechniques, see Aslam and Dent (Aslam and Dent, “Bioconjugation: ProteinCoupling Techniques for the Biomedical Sciences,” Macmillan ReferenceLtd., London, England, 1998), incorporated hereinafter by reference inits entirety.

In a specific embodiment, antibodies to a CVHPP may be used to eliminatethe CVHPP in vivo by activating the complement system or mediatingantibody-dependent cellular cytotoxicity (ADCC), or cause uptake of theantibody coated cells by the receptor-mediated endocytosis (RE) system.

Vectors

Another aspect of the invention pertains to vectors containing apolynucleotide encoding a CVHPP, a variant of a CVHPP, or a portionthereof. One type of vector is a “plasmid,” which includes a circulardouble-stranded DNA loop into which additional DNA segments can beligated. In the present specification, “plasmid” and “vector” can beused interchangeably as the plasmid is the most commonly used form ofvector. Vectors also include expression vectors and gene deliveryvectors.

The expression vectors of the invention comprise a polynucleotideencoding a CVHPP or a portion thereof in a form suitable for expressionof the polynucleotide in a host cell, which means that the expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, and operatively linked tothe polynucleotide sequence to be expressed. It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, such as CVHPPs, mutant forms of CVHPPs,CVHPP-fusion proteins, and the like.

The expression vectors of the invention can be designed for expressionof CVHPPs in prokaryotic or eukaryotic cells. For example, CVHPPs can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors), yeast cells or mammalian cells.Alternatively, the expression vector can be transcribed and translatedin vitro, for example using T7 promoter regulatory sequences and T7polymerase.

The expression of proteins in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression of therecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia, Piscataway,N.J.), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia,Piscataway, N.J.) which fuse glutathione S transferase (GST), maltose Ebinding protein, and protein A, respectively, to the target recombinantprotein.

Purified fusion proteins can be utilized in CVHPP activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for CVHPPs.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. Another strategy is toalter the polynucleotide sequence of the polynucleotide to be insertedinto an expression vector so that the individual codons for each aminoacid are those preferentially utilized in E. coli. Such alteration ofpolynucleotide sequences of the invention can be carried out by standardDNA synthesis techniques.

In another embodiment, the CVHPP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1, pMFa, pJRY88, pYES2 and picZ (Invitrogen Corp, SanDiego, Calif.).

Alternatively, CVHPPs of the invention can be expressed in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series and the pVL series.

In yet another embodiment, a polynucleotide of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 and pMT2PC. Whenused in mammalian cells, the expression vector's control functions areoften provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2 and 5, cytomegalovirusand Simian Virus 40. Target gene expression from the pTrc vector relieson host RNA polymerase transcription from a hybrid trp-lac fusionpromoter. Target gene expression from the pET 11d vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HSLE174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

In another embodiment, the mammalian expression vector is capable ofdirecting expression of the polynucleotide preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the polynucleotide). Tissue-specific regulatory elements areknown in the art and may include epithelial cell-specific promoters.Other non-limiting examples of suitable tissue-specific promotersinclude the liver-specific promoter (e.g., albumin promoter),lymphoid-specific promoters, promoters of T cell receptors andimmunoglobulins, neuron-specific promoters (e.g., the neurofilamentpromoter), pancreas-specific promoters (e.g., insulin promoter), andmammary gland-specific promoters (e.g., milk whey promoter).Developmentally-regulated promoters (e.g., the α-fetoprotein promoter)are also encompassed.

The invention provides a recombinant expression vector comprising apolynucleotide encoding a CVHPP cloned into the expression vector in anantisense orientation. That is, the DNA molecule is operatively linkedto a regulatory sequence in a manner which allows for expression (bytranscription of the DNA molecule) of an RNA molecule which is antisenseto mRNA corresponding to a CVHG of the invention. Regulatory sequencesoperatively linked to a polynucleotide cloned in the antisenseorientation can be chosen which direct the continuous expression of theantisense RNA molecule in a variety of cell types, for instance, viralpromoters and/or enhancers, or regulatory sequences can be chosen whichdirect constitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensepolynucleotides are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced.

The invention further provides gene delivery vehicles for delivery ofpolynucleotides to cells, tissues, or a mammal for expression. Forexample, a polynucleotide sequence of the invention can be administeredeither locally or systemically in a gene delivery vehicle. Theseconstructs can utilize viral or non-viral vector approaches in in vivoor ex vivo modality. Expression of the coding sequence can be inducedusing endogenous mammalian or heterologous promoters. Expression of thecoding sequence in vivo can be either constituted or regulated. Theinvention includes gene delivery vehicles capable of expressing thecontemplated polynucleotides. The gene delivery vehicle is preferably aviral vector and, more preferably, a retroviral, lentiviral, adenoviral,adeno-associated viral (AAV), herpes viral, or alphavirus vector. Theviral vector can also be an astrovirus, coronavirus, orthomyxovirus,papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus,togavirus viral vector.

The delivery of gene therapy constructs of this invention into cells isnot limited to the above mentioned viral vectors. Other delivery methodsand media may be employed such as, for example, nucleic acid expressionvectors, polycationic condensed DNA linked or unlinked to killedadenovirus alone, ligand linked DNA, liposomes, eukaryotic cell deliveryvehicles cells, deposition of photopolymerized hydrogel materials,handheld gene transfer particle gun, ionizing radiation, nucleic chargeneutralization or fusion with cell membranes. Particle mediated genetransfer may be employed. Briefly, DNA sequence can be inserted intoconventional vectors that contain conventional control sequences forhigh level expression, and then be incubated with synthetic genetransfer molecules such as polymeric DNA-binding cations likepolylysine, protamine, and albumin, linked to cell targeting ligandssuch as asialoorosomucoid, insulin, galactose, lactose or transferrin.Naked DNA may also be employed. Uptake efficiency of naked DNA may beimproved using biodegradable latex beads. The method may be improvedfurther by treatment of the beads to increase hydrophobicity and therebyfacilitate disruption of the endosome and release of the DNA into thecytoplasm.

Another aspect of the invention pertains to the expression of CVHPPsusing a regulatable expression system. Systems to regulate expression oftherapeutic genes have been developed and incorporated into the currentviral and nonviral gene delivery vectors. These systems are brieflydescribed below:

Tet-on/off system. The Tet-system is based on two regulatory elementsderived from the tetracycline-resistance operon of the E. coli Tn10transposon: the tet repressor protein (TetR) and the Tet operator DNAsequence (tetO) to which TetR binds. The system consists of twocomponents, a “regulator” and a “reporter” plasmid. The “regulator”plasmid encodes a hybrid protein containing a mutated Tet repressor(rtetR) fused to the VP16 activation domain of herpes simplex virus. The“reporter” plasmid contains a tet-responsive element (TRE), whichcontrols the “reporter” gene of choice. The rtetR-VP16 fusion proteincan only bind to the TRE, therefore activates the transcription of the“reporter” gene, in the presence of tetracycline. The system has beenincorporated into a number of viral vectors including retrovirus,adenovirus and AAV.

Ecdysone system. The ecdysone system is based on the molting inductionsystem found in Drosophila, but modified for inducible expression inmammalian cells. The system uses an analog of the drosophila steroidhormone ecdysone, muristerone A, to activate expression of the gene ofinterest via a heterodimeric nuclear receptor. Expression levels havebeen reported to exceed 200-fold over basal levels with no effect onmammalian cell physiology.

Progesterone system. The progesterone receptor is normally stimulated tobind to a specific DNA sequence and to activate transcription through aninteraction with its hormone ligand. Conversely, the progesteroneantagonist mifepristone (RU486) is able to block hormone-induced nucleartransport and subsequent DNA binding. A mutant form of the progesteronereceptor that can be stimulated to bind through an interaction withRU486 has been generated. To generate a specific, regulatabletranscription factor, the RU486-binding domain of the progesteronereceptor has been fused to the DNA-binding domain of the yeasttranscription factor GAL4 and the transactivation domain of the HSVprotein VP16. The chimeric factor is inactive in the absence of RU486.The addition of hormone, however, induces a conformational change in thechimeric protein, and this change allows binding to a GAL4-binding siteand the activation of transcription from promoters containing theGAL4-binding site.

Rapamycin system. Immunosuppressive agents, such as FK506 and rapamycin,act by binding to specific cellular proteins and facilitating theirdimerization. For example, the binding of rapamycin to FK506-bindingprotein (FKBP) results in its heterodimerization with another rapamycinbinding protein FRAP, which can be reversed by removal of the drug. Theability to bring two proteins together by addition of a drug potentiatesthe regulation of a number of biological processes, includingtranscription. A chimeric DNA-binding domain has been fused to the FKBP,which enables binding of the fusion protein to a specific DNA-bindingsequence. A transcriptional activation domain also has been fused toFRAP. When these two fusion proteins are co-expressed in the same cell,a fully functional transcription factor can be formed byheterodimerization mediated by addition of rapamycin. The dimerizedchimeric transcription factor can then bind to a synthetic promotersequence containing copies of the synthetic DNA-binding sequence. Thissystem has been successfully integrated into adenoviral and AAV vectors.Long term regulatable gene expression has been achieved in both mice andbaboons.

Immunogens and Immunogenic Compositions

Within certain aspects, CVHPP, CVHPN, CVHPP-specific T cell,CVHPP-presenting APC, CVHG-containing vectors, including but are notlimited to expression vectors and gene delivery vectors, may be utilizedas vaccines for CVH. Vaccines may comprise one or more suchcompounds/cells and an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated). Vaccines within the scope of the present invention mayalso contain other compounds, which may be biologically active orinactive. For example, one or more immunogenic portions of otherantigens may be present, either incorporated into a fusion polypeptideor as a separate compound, within the composition of vaccine.

A vaccine may contain DNA encoding one or more CVHPP or portion ofCVHPP, such that the polypeptide is generated in situ. As noted above,the DNA may be present within any of a variety of delivery systems knownto those of ordinary skill in the art, including nucleic acid expressionvectors, gene delivery vectors, and bacteria expression systems.Numerous gene delivery techniques are well known in the art. Appropriatenucleic acid expression systems contain the necessary DNA sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). Bacterial delivery systems involve the administration of abacterium (such as Bacillus-Calmette-Guerrin) that expresses animmunogenic portion of the polypeptide on its cell surface or secretessuch an epitope. In a preferred embodiment, the DNA may be introducedusing a viral expression system (e.g., vaccinia or other pox virus,retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA may also be “naked,” as described,for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewedby Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may beincreased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells. It will be apparent that avaccine may comprise both a polynucleotide and a polypeptide component.Such vaccines may provide for an enhanced immune response.

It will be apparent that a vaccine may contain pharmaceuticallyacceptable salts of the polynucleotides and polypeptides providedherein. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases, including organic bases (e.g., salts of primary,secondary and tertiary amines and basic amino acids) and inorganic bases(e.g., sodium, potassium, lithium, ammonium, calcium and magnesiumsalts).

Any of a variety of immunostimulants may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. As definedpreviously, an “adjuvant” is a substance that serves to enhance theimmunogenicity of an antigen. Thus, adjuvants are often given to boostthe immune response and are well known to the skilled artisan. Examplesof adjuvants contemplated in the present invention include, but are notlimited to, aluminum salts (alum) such as aluminum phosphate andaluminum hydroxide, Mycobacterium tuberculosis, Bordetella pertussis,bacterial lipopolysaccharides, aminoalkyl glucosamine phosphatecompounds (AGP), or derivatives or analogs thereof, which are availablefrom Corixa (Hamilton, Mont.), and which are described in U.S. Pat. No.6,113,918; one such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyoxytetradecanoylamino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion, MPL™(3-O-deacylated monophosphoryl lipid A) (Corixa) described in U.S. Pat.No. 4,912,094, synthetic polynucleotides such as oligonucleotidescontaining a CpG motif (U.S. Pat. No. 6,207,646), polypeptides, saponinssuch as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, Mass.),described in U.S. Pat. No. 5,057,540, a pertussis toxin (PT), or an E.coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109,PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302and WO 92/19265, cholera toxin (either in a wild-type or mutant form,e.g., wherein the glutamic acid at amino acid position 29 is replaced byanother amino acid, preferably a histidine, in accordance with publishedInternational Patent Application number WO 00/18434). Various cytokinesand lymphokines are suitable for use as adjuvants. One such adjuvant isgranulocyte-macrophage colony stimulating factor (GM-CSF), which has anucleotide sequence as described in U.S. Pat. No. 5,078,996. A plasmidcontaining GM-CSF cDNA has been transformed into E. coli and has beendeposited with the American Type Culture Collection (ATCC), 1081University Boulevard, Manassas, Va. 20110-2209, under Accession Number39900. The cytokine IL-12 is another adjuvant which is described in U.S.Pat. No. 5,723,127. Other cytokines or lymphokines have been shown tohave immune modulating activity, including, but not limited to, theinterleukins 1-alpha, 1-beta, 2, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16, 17and 18, the interferons-alpha, beta and gamma, granulocyte colonystimulating factor, and the tumor necrosis factors alpha and beta, andare suitable for use as adjuvants.

Any vaccine provided herein may be prepared using well known methodsthat result in a combination of antigen, immune response enhancer and asuitable carrier or excipient. The compositions described herein may beadministered as part of a sustained release formulation (i.e., aformulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology and administered by, for example, oral,rectal or subcutaneous implantation, or by implantation at the desiredtarget site. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), as well as polyacrylate,latex, starch, cellulose and dextran. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

Any of a variety of delivery vehicles may be employed within vaccines tofacilitate production of an antigen-specific immune response thattargets cancer cells. Delivery vehicles include antigen presenting cells(APCs), such as dendritic cells, macrophages, B cells, monocytes andother cells that may be engineered to be efficient APCs. Such cells may,but need not, be genetically modified to increase the capacity forpresenting the antigen, to improve activation and/or maintenance of theT cell response, to have anti-CVH effects per se and/or to beimmunologically compatible with the receiver (i.e., matched HLAhaplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, and may be autologous, allogeneic,syngeneic or xenogenic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as APCs. Dendritic cells are highly potentAPCs and have been shown to be effective as a physiological adjuvant foreliciting prophylactic or therapeutic anti-CVH immunity. In general,dendritic cells may be identified based on their typical shape (stellatein situ, with marked cytoplasmic processes (dendrites) visible invitro), their ability to take up, process and present antigens with highefficiency and their ability to activate naive T cell responses.Dendritic cells may, of course, be engineered to express specificcell-surface receptors or ligands that are not commonly found ondendritic cells in vivo, and such modified dendritic cells arecontemplated by the present invention. As an alternative to dendriticcells, secreted vesicles antigen-loaded dendritic cells (calledexosomes) may be used within a vaccine (see Zitvogel et al., Nature Med.4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, lymph nodes, spleen, skin, umbilical cord blood or anyother suitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcy receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding a CVHPP(or portion or other variant thereof) such that the CVHPP, or animmunogenic portion thereof, is expressed on the cell surface. Suchtransfection may take place ex vivo, and a composition or vaccinecomprising such transfected cells may then be used for therapeuticpurposes, as described herein. Alternatively, a gene delivery vehiclethat targets a dendritic or other antigen presenting cell may beadministered to a patient, resulting in transfection that occurs invivo. In vivo and ex vivo transfection of dendritic cells, for example,may generally be performed using any methods known in the art, such asthose described in WO 97/24447, or the gene gun approach described byMahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigenloading of dendritic cells may be achieved by incubating dendritic cellsor progenitor cells with the CVHPP, DNA (naked or within a plasmidvector) or RNA; or with antigen-expressing recombinant bacterium orviruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).Prior to loading, the polypeptide may be covalently conjugated to animmunological partner that provides T cell help (e.g., a carriermolecule). Alternatively, a dendritic cell may be pulsed with anon-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Vaccines may be presented in unit-dose or multi-dose containers, such assealed ampoules or vials. Such containers are preferably hermeticallysealed to preserve sterility of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a vaccine may be stored in afreeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

Detection Methods

As discussed earlier, expression level of CVHGs may be used as a markerfor CVH and CVH-related disorders. Detection and measurement of therelative amount of a CVHG product (polynucleotide or polypeptide) of theinvention can be by any method known in the art. Typical methodologiesfor detection of a transcribed polynucleotide include RNA extractionfrom a cell or tissue sample, followed by hybridization of a labeledprobe (i.e., a complementary polynucleotide molecule) specific for thetarget RNA to the extracted RNA and detection of the probe (i.e.,Northern blotting).

Typical methodologies for peptide detection include protein extractionfrom a cell or tissue sample, followed by binding of an antibodyspecific for the target protein to the protein sample, and detection ofthe antibody. For example, detection of desmin may be accomplished usingpolyclonal antibody anti-desmin. Antibodies are generally detected bythe use of a labeled secondary antibody. The label can be aradioisotope, a fluorescent compound, an enzyme, an enzyme co-factor, orligand. Such methods are well understood in the art.

In certain embodiments, the CVHGs themselves (i.e., the DNA or cDNA) mayserve as markers for CVH. For example, an increase of genomic copies ofa CVHG, such as by duplication of the gene, may also be correlated withCVH and CVH-associated disorders.

Detection of specific polynucleotide molecules may also be assessed bygel electrophoresis, column chromatography, or direct sequencing,quantitative PCR (in the case of polynucleotide molecules), RT-PCR, ornested-PCR among many other techniques well known to those skilled inthe art.

Detection of the presence or number of copies of all or a part of anCVHG of the invention may be performed using any method known in theart. Typically, it is convenient to assess the presence and/or quantityof a DNA or cDNA by Southern analysis, in which total DNA from a cell ortissue sample is extracted and hybridized with a labeled probe (i.e., acomplementary DNA molecules). The probe is then detected and quantified.The label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Other useful methods of DNA detectionand/or quantification include direct sequencing, gel electrophoresis,column chromatography, and quantitative PCR, as is known by one skilledin the art.

In certain embodiments, CVHPPs may serve as markers for CVH. Detectionof specific polypeptide molecules may be assessed by gelelectrophoresis, Western blot, column chromatography, or directsequencing, among many other techniques well known to those skilled inthe art.

Panels of CVHGs

The expression level of each CVHG may be considered individually,although it is within the scope of the invention to provide combinationsof two or more CVHGs for use in the methods and compositions of theinvention to increase the confidence of the analysis. In another aspect,the invention provides panels of the CVHGs of the invention. A panel ofCVHGs comprises two or more CVHGs. A panel may also comprise 2-5, 5-15,15-35, 35-50, or more than 50 CVHGs. In a preferred embodiment, thesepanels of CVHGs are selected such that the CVHGs within any one panelshare certain features. For example, the CVHGs of a first panel may allrelate to myelination in a CVH sample. Alternatively, CVHGs of a secondpanel may each exhibit differential regulation as compared to a firstpanel. Similarly, different panels of CVHGs may be composed of CVHGsrepresenting different stages of CVH. Panels of the CVHGs of theinvention may be made by independently selecting CVHGs from Tables 3-8,and may further be provided on biochips, as discussed below.

Screening Methods

The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents comprising therapeutic moieties (e.g., peptides,peptidomimetics, peptoids, polynucleotides, small molecules or otherdrugs) which (a) bind to a CVHPP, or (b) have a modulatory (e.g.,stimulatory or inhibitory) effect on the activity of a CVHPP or, morespecifically, (c) have a modulatory effect on the interactions of theCVHPP with one or more of its natural substrates (e.g., peptide,protein, hormone, co-factor, or polynucleotide), or (d) have amodulatory effect on the expression of the CVHPPs. Such assays typicallycomprise a reaction between the CVHPP and one or more assay components.The other components may be either the test compound itself, or acombination of the test compound and a binding partner of the CVHPP.

To screen for compounds which interfere with binding of two proteinse.g., a CVHPP and its binding partner, a Scintillation Proximity Assaycan used. In this assay, the CVHPP is labeled with an isotope such as¹²⁵I. The binding partner is labeled with a scintillant, which emitslight when proximal to radioactive decay (i.e., when the CVHPP is boundto its binding partner). A reduction in light emission will indicatethat a compound has interfered with the binding of the two proteins.

Alternatively a Fluorescence Energy Transfer (FRET) assay could be used.In a FRET assay of the invention, a fluorescence energy donor iscomprised on one protein (e.g., a CVHPP) and a fluorescence energyacceptor is comprised on a second protein (e.g., a binding partner ofthe CVHPP). If the absorption spectrum of the acceptor molecule overlapswith the emission spectrum of the donor fluorophore, the fluorescentlight emitted by the donor is absorbed by the acceptor. The donormolecule can be a fluorescent residue on the protein (e.g., intrinsicfluorescence such as a tryptophan or tyrosine residue), or a fluorophorewhich is covalently conjugated to the protein (e.g., fluoresceinisothiocyanate, FITC). An appropriate donor molecule is then selectedwith the above acceptor/donor spectral requirements in mind.

Thus, in this example, a CVHPP is labeled with a fluorescent molecule(i.e., a donor fluorophore) and its binding partner is labeled with aquenching molecule (i.e., an acceptor). When the CVHPP and its bindingpartner are bound, fluorescence emission will be quenched or reducedrelative the CVHPP alone. Similarly, a compound which can dissociate theinteraction of the CVHPP-partner complex, will result in an increase influorescence emission, which indicates the compound has interfered withthe binding of the CVHPP to its binding partner.

Another assay to detect binding or dissociation of two proteins isfluorescence polarization or anisotropy. In this assay, the investigatedprotein (e.g., a CVHPP) is labeled with a fluorophore with anappropriate fluorescence lifetime. The protein sample is then excitedwith vertically polarized light. The value of anisotropy is thencalculated by determining the intensity of the horizontally andvertically polarized emission light. Next, the labeled protein (CVHPP)is mixed with a CVHPP binding partner and the anisotropy measured again.Because fluorescence anisotropy intensity is related to the rotationalfreedom of the labeled protein, the more rapidly a protein rotates insolution, the smaller the anisotropy value. Thus, if the labeled CVHPPis part of a complex (e.g., CVHPP-partner), the CVHPP rotates moreslowly in solution (relative to free, unbound CVHPP) and the anisotropyintensity increases. Subsequently, a compound which can dissociate theinteraction of the CVHPP-partner complex, will result in a decrease inanisotropy (i.e., the labeled CVHPP rotates more rapidly), whichindicates the compound has interfered with the binding of CVHPP to itsbinding partner.

A more traditional assay would involve labeling the CVHPP bindingpartner with an isotope such as ¹²⁵I, incubating with the CVHPP, thenimmunoprecipitating of the CVHPP. Compounds that increase the free CVHPPwill decrease the precipitated counts. To avoid using radioactivity, theCVHPP binding partner could be labeled with an enzyme-conjugatedantibody instead.

Alternatively, the CVHPP binding partner could be immobilized on thesurface of an assay plate and the CVHPP could be labeled with aradioactive tag. A rise in the number of counts would identify compoundsthat had interfered with binding of the CVHPP and its binding partner.

Evaluation of binding interactions may further be performed usingBiacore technology, wherein the CVHPP or its binding partner is bound toa micro chip, either directly by chemical modification or tethered viaantibody-epitope association (e.g., antibody to the CVHPP), antibodydirected to an epitope tag (e.g., His tagged) or fusion protein (e.g.,GST). A second protein or proteins is/are then applied via flow over the“chip” and the change in signal is detected. Finally, test compounds areapplied via flow over the “chip” and the change in signal is detected.

Once a series of potential compounds has been identified for acombination of CVHPP, CVHPP binding partner and ALS, a bioassay can beused to select the most promising candidates. For example, a cellularassay that measures cell proliferation in presence of the CVHPP and theCVHPP binding partner was described above. This assay could be modifiedto test the effectiveness of small molecules that interfere with bindingof a CVHPP and its binding partner in enhancing cellular proliferation.An increase in cell proliferation would correlate with a compound'spotency.

The test compounds of the present invention are generally either smallmolecules or biomolecules. Small molecules include, but are not limitedto, inorganic molecules and small organic molecules. Biomoleculesinclude, but are not limited to, naturally-occurring and syntheticcompounds that have a bioactivity in mammals, such as lipids, steroids,polypeptides, polysaccharides, and polynucleotides. In one preferredembodiment, the test compound is a small molecule. In another preferredembodiment, the test compound is a biomolecule. One skilled in the artwill appreciate that the nature of the test compound may vary dependingon the nature of the CVHPP. For example, if the CVHPP is an orphanreceptor having an unknown ligand, the test compound may be any of anumber of biomolecules which may act as cognate ligand, including butnot limited to, cytokines, lipid-derived mediators, small biogenicamines, hormones, neuropeptides, or proteases.

The test compounds of the present invention may be obtained from anyavailable source, including systematic libraries of natural and/orsynthetic compounds. Test compounds may also be obtained by any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive); spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library and peptoid library approaches are limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds. As usedherein, the term “binding partner” refers to a molecule which serves aseither a substrate for a CVHPP, or alternatively, as a ligand havingbinding affinity to the CVHPP.

Screening for Inhibitors of CVHPP

The invention provides methods of screening test compounds forinhibitors of CVHPP. The method of screening comprises obtaining samplesfrom subjects diagnosed with or suspected of having CVH, contacting eachseparate aliquot of the samples with one of a plurality of testcompounds, and comparing expression of one or more CVHGs in each of thealiquots to determine whether any of the test compounds provides asubstantially altered level of expression or activity of an CVHGrelative to samples with other test compounds or relative to anuntreated sample or control sample. In addition, methods of screeningmay be devised by combining a test compound with a protein and therebydetermining the effect of the test compound on the protein.

In addition, the invention is further directed to a method of screeningfor test compounds capable of modulating with the binding of a CVHPP anda binding partner, by combining the test compound, CVHPP, and bindingpartner together and determining whether binding of the binding partnerand CVHPP occurs. The test compound may be either small molecules or abioactive agent. As discussed below, test compounds may be provided froma variety of libraries well known in the art.

Modulators of CVHG expression, activity or binding ability are useful astherapeutic compositions of the invention. Such modulators (e.g.,antagonists or agonists) may be formulated as pharmaceuticalcompositions, as described herein below. Such modulators may also beused in the methods of the invention, for example, to diagnose, treat,or prognose CVH.

High-Throughput Screening Assays

The invention provides methods of conducting high-throughput screeningfor test compounds capable of inhibiting activity or expression of aCVHPP of the present invention. In one embodiment, the method ofhigh-throughput screening involves combining test compounds and theCVHPP and detecting the effect of the test compound on the CVHPP.

A variety of high-throughput functional assays well-known in the art maybe used in combination to screen and/or study the reactivity ofdifferent types of activating test compounds. Since the coupling systemis often difficult to predict, a number of assays may need to beconfigured to detect a wide range of coupling mechanisms. A variety offluorescence-based techniques are well-known in the art and are capableof high-throughput and ultra high throughput screening for activity,including but not limited to BRET® or FRET® (both by Packard InstrumentCo., Meriden, Conn.). The ability to screen a large volume and a varietyof test compounds with great sensitivity permits analysis of thetherapeutic targets of the invention to further provide potentialinhibitors of CVH. For example, where the CVHG encodes an orphanreceptor with an unidentified ligand, high-throughput assays may beutilized to identify the ligand, and to further identify test compoundswhich prevent binding of the receptor to the ligand. The BIACORE® systemmay also be manipulated to detect binding of test compounds withindividual components of the therapeutic target, to detect binding toeither the encoded protein or to the ligand.

By combining test compounds with CVHPPs of the invention and determiningthe binding activity between them, diagnostic analysis can be performedto elucidate the coupling systems. Generic assays using cytosensormicrophysiometer may also be used to measure metabolic activation, whilechanges in calcium mobilization can be detected by using thefluorescence-based techniques such as FLIPR® (Molecular Devices Corp,Sunnyvale, Calif.). In addition, the presence of apoptotic cells may bedetermined by TUNEL assay, which utilizes flow cytometry to detect free3-OH termini resulting from cleavage of genomic DNA during apoptosis. Asmentioned above, a variety of functional assays well-known in the artmay be used in combination to screen and/or study the reactivity ofdifferent types of activating test compounds. Preferably, thehigh-throughput screening assay of the present invention utilizeslabel-free plasmon resonance technology as provided by BIACORE® systems(Biacore International AB, Uppsala, Sweden). Plasmon free resonanceoccurs when surface plasmon waves are excited at a metal/liquidinterface. By reflecting directed light from the surface as a result ofcontact with a sample, the surface plasmon resonance causes a change inthe refractive index at the surface layer. The refractive index changefor a given change of mass concentration at the surface layer is similarfor many bioactive agents (including proteins, peptides, lipids andpolynucleotides), and since the BIACORE® sensor surface can befunctionalized to bind a variety of these bioactive agents, detection ofa wide selection of test compounds can thus be accomplished.

Therefore, the invention provides for high-throughput screening of testcompounds for the ability to inhibit activity of a protein encoded bythe CVHGs listed in Table 4, by combining the test compounds and theprotein in high-throughput assays such as BIACORE®, or influorescence-based assays such as BRET®. In addition, high-throughputassays may be utilized to identify specific factors which bind to theencoded proteins, or alternatively, to identify test compounds whichprevent binding of the receptor to the binding partner. In the case oforphan receptors, the binding partner may be the natural ligand for thereceptor. Moreover, the high-throughput screening assays may be modifiedto determine whether test compounds can bind to either the encodedprotein or to the binding partner (e.g., substrate or ligand) whichbinds to the protein.

Predictive Medicine

The present invention pertains to the field of predictive medicine inwhich diagnostic assays, prognostic assays, pharmacogenetics andmonitoring clinical trials are used for prognostic (predictive) purposeto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningCVHG expression and/or activity, in the context of a biological sample(e.g., blood, urine, feces, serum, cells, tissue) to thereby determinewhether an individual is at risk for developing CVH associated withaltered CVHG expression or activity. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing CVH associated with aberrant CVHG expression oractivity.

For example, the number of copies of an CVHG can be assayed in abiological sample. Such assays can be used for prognostic or predictivepurposes to thereby prophylactically treat an individual prior to theonset of CVH associated with aberrant CVHG protein, polynucleotideexpression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of CVHGsin clinical trials.

Diagnostic Assays

An exemplary method for detecting the presence or absence of a CVHPP orCVHPN in a biological sample involves contacting a biological samplewith a compound or an agent capable of detecting the CVHPP or CVHPN(e.g., mRNA, genomic DNA). A preferred agent for detecting mRNA orgenomic DNA corresponding to an CVHG or CVHPP of the invention is alabeled polynucleotide probe capable of hybridizing to a mRNA or genomicDNA of the invention. In a most preferred embodiment, thepolynucleotides to be screened are arranged on a GeneChip®. Suitableprobes for use in the diagnostic assays of the invention are describedherein. A preferred agent for detecting a CVHPP of the invention is anantibody which specifically recognizes the CVHPP.

The diagnostic assays may also be used to quantify the amount ofexpression or activity of a CVHG in a biological sample. Suchquantification is useful, for example, to determine the progression orseverity of CVH and CVH-related disorders. Such quantification is alsouseful, for example, to determine the severity of CVH followingtreatment.

Determining Severity of CVH and CVH-Related Diseases

In the field of diagnostic assays, the invention also provides methodsfor determining the severity of CVH by isolating a sample from a subject(e.g., a colon biopsy), and detecting the presence, quantity and/oractivity of one or more CVHG products in the sample relative to a secondsample from a normal sample or control sample. In one embodiment, theexpression levels of CVHGs in the two samples are compared, and amodulation in one or more CVHGs in the test sample indicates CVH. Inother embodiments the modulation of 2, 3, 4 or more CVHGs indicates asevere case of CVH.

In another aspect, the invention provides CVHG products whose quantityor activity is correlated with the severity of CVH. The subsequent levelof expression may further be compared to different expression profilesof various stages of the disease to confirm whether the subject has amatching profile. In yet another aspect, the invention provides CVHGswhose quantity or activity is correlated with a risk in a subject fordeveloping CVH.

A preferred agent for detecting a CVHPP is an antibody capable ofbinding to the CVHPP, preferably an antibody with a detectable label.Antibodies can be polyclonal or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled,” with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect CVHG mRNA, protein or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of CVHG mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of CVHPP includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of CVHG genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of CVHPP includeintroducing into a subject a labeled anti-CVHPP antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a tissue or serum sampleisolated by conventional means from a subject, e.g., a biopsy or blooddraw.

Prognostic Assays

The diagnostic method described herein can be utilized to identifysubjects having or at risk of developing CVH associated with aberrantCVHG expression or activity.

The assays described herein, such as the preceding or following assays,can be utilized to identify a subject having CVH associated with anaberrant level of CVHG activity or expression. Alternatively, theprognostic assays can be utilized to identify a subject at risk fordeveloping CVH associated with aberrant levels of CVHG protein activityor polynucleotide expression. Thus, the present invention provides amethod for identifying CVH associated with aberrant CVHG expression oractivity in which a test sample is obtained from a subject and CVHPP orCVHPN (e.g., mRNA or genomic DNA) is detected, wherein the presence ofCVHPP or CVHPN is diagnostic or prognostic for a subject having or atrisk of developing CVH with aberrant CVHG expression or activity.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, polynucleotide,small molecule, or other drug candidate) to treat or prevent CVHassociated with aberrant CVHG expression or activity, such as, forexample, a cytokine. For example, such methods can be used to determinewhether a subject can be effectively treated with an agent to inhibitCVH. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for CVHassociated with aberrant CVHG expression or activity.

Prognostic assays can be devised to determine whether a subjectundergoing treatment for CVH has a poor outlook for disease progression.In a preferred embodiment, prognosis can be determined shortly afterdiagnosis, i.e., within a few days. By establishing expression profilesof different stages of CVHGs, from onset to later stages, an expressionpattern may emerge to correlate a particular expression profile toincreased likelihood of a poor prognosis. The prognosis may then be usedto devise a more aggressive treatment program and enhance the likelihoodof success.

The methods of the invention can also be used to detect geneticalterations in a CVHG, thereby determining if a subject with the alteredgene is at risk for damage characterized by aberrant regulation in CVHGexpression or activity. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic alteration characterized by at least one alterationaffecting the integrity of a CVHG, or the aberrant expression of theCVHG. For example, such genetic alterations can be detected byascertaining the existence of at least one of the following: 1) deletionof one or more nucleotides from a CVHG; 2) addition of one or morenucleotides to a CVHG; 3) substitution of one or more nucleotides of aCVHG, 4) a chromosomal rearrangement of a CVHG; 5) alteration in thelevel of a messenger RNA transcript of a CVHG, 6) aberrant modificationof a CVHG, such as of the methylation pattern of the genomic DNA, 7) thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of a CVHG, 8) non-wild type level of a CVHPP, 9) allelic lossof a CVHG, and 10) inappropriate post-translational modification of aCVHPP. As described herein, there are a large number of assays known inthe art, which can be used for detecting alterations in a CVHG or a CVHGproduct. A preferred biological sample is a blood sample isolated byconventional means from a subject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR), thelatter of which can be particularly useful for detecting point mutationsin the CVHG. This method can include the steps of collecting a sample ofcells from a subject, isolating a polynucleotide sample (e.g., genomic,mRNA or both) from the cells of the sample, contacting thepolynucleotide sample with one or more primers which specificallyhybridize to a CVHG under conditions such that hybridization andamplification of the CVHG (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. It is understood that PCR and/or LCR may be desirable to be usedas a preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include: self-sustained sequencereplication, transcriptional amplification system, Q-Beta Replicase, orany other polynucleotide amplification method, followed by the detectionof the amplified molecules using techniques well known to those of skillin the art. These detection schemes are especially useful for thedetection of polynucleotide molecules if such molecules are present invery low numbers.

In an alternative embodiment, mutations in an CVHG from a sample cellcan be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicate mutations in the sample DNA. Moreover, sequencespecific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations in a CVHG can be identified byhybridizing sample and control polynucleotides, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes. For example, genetic mutations in a CVHG can be identified intwo dimensional arrays containing light generated DNA probes. Briefly, afirst hybridization array of probes can be used to scan through longstretches of DNA in a sample and control to identify base changesbetween the sequences by making linear arrays of sequential overlappingprobes. This step allows the identification of point mutations. Thisstep is followed by a second hybridization array that allows thecharacterization of specific mutations by using smaller, specializedprobe arrays complementary to all variants or mutations detected. Eachmutation array is composed of parallel probe sets, one complementary tothe wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the CVHG and detectmutations by comparing the sequence of the sample CVHG with thecorresponding wild-type (control) sequence. It is also contemplated thatany of a variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays, including sequencing by massspectrometry.

Other methods for detecting mutations in a CVHG include methods in whichprotection from cleavage agents is used to detect mismatched bases inRNA/RNA or RNA/DNA heteroduplexes. In general, the art technique of“mismatch cleavage” starts by providing heteroduplexes by hybridizing(labeled) RNA or DNA containing the wild-type CVHG sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex, which will exist due to basepairmismatches between the control and sample strands. For instance, RNA/DNAduplexes can be treated with RNase and DNA/DNA hybrids treated with S1nuclease to enzymatically digest the mismatched regions. In otherembodiments, either DNA/DNA or RNA/DNA duplexes can be treated withhydroxylamine or osmium tetroxide and with piperidine in order to digestmismatched regions. After digestion of the mismatched regions, theresulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. In a preferredembodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in CVHG cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches. According to an exemplary embodiment, a probe basedon an CVHG sequence, e.g., a wild-type CVHG sequence, is hybridized tocDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, forexample, U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in CVHGs. For example, single-strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type polynucleotides.Single-stranded DNA fragments of sample and control CVHG polynucleotideswill be denatured and allowed to renature. The secondary structure ofsingle-stranded polynucleotides varies according to sequence. Theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA) in which the secondary structureis more sensitive to a change in sequence. In a preferred embodiment,the subject method utilizes heteroduplex analysis to separatedouble-stranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. Trends Genet. 7:5, 1991).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE). When DGGE is usedas the method of analysis, DNA will be modified to insure that it doesnot completely denature, for example, by adding a GC clamp ofapproximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturinggradient to identify differences in the mobility of control and sampleDNA (Rosenbaum and Reissner Biophys Chem 265:12753, 1987).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. Proc. Natl. Acad. Sci. USA 86:6230, 1989). Such allele specificoligonucleotides are hybridized to PCR amplified target or a number ofdifferent mutations when the oligonucleotides are attached to thehybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) or at theextreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent or reduce polymerase extension. In addition, it maybe desirable to introduce a novel restriction site in the region of themutation to create cleavage-based detection. It is anticipated that, incertain embodiments, amplification may also be performed using Taqligase for amplification. In such cases, ligation will occur only ifthere is a perfect match at the 3′ end of the 5′ sequence, thus makingit possible to detect the presence of a known mutation at a specificsite by looking for the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits comprising at least one probe polynucleotideor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose subjects exhibiting symptoms orfamily history of a disease or illness involving a CVHG.

Furthermore, any cell type or tissue in which a CVHG is expressed may beutilized in the prognostic or diagnostic assays described herein.

Monitoring Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, small molecules,proteins, nucleotides) on the expression or activity of a CVHPP can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay, as described herein to decrease CVHG expression or activity, canbe monitored in clinical trials of subjects exhibiting increased CVHGexpression or activity. In such clinical trials, the expression oractivity of an CVHG can be used as a “read-out” of the phenotype of aparticular tissue.

For example, and not by way of limitation, CVHGs that are modulated intissues by treatment with an agent can be identified. Thus, to study theeffect of agents on the CVHPP in a clinical trial, cells can be isolatedand RNA prepared and analyzed for the levels of expression of an CVHG.The levels of gene expression or a gene expression pattern can bequantified by Northern blot analysis, RT-PCR or GeneChip® as describedherein, or alternatively by measuring the amount of protein produced, byone of the methods as described herein, or by measuring the levels ofactivity of CVHPP. In this way, the gene expression pattern can serve asa read-out, indicative of the physiological response of the cells to theagent. Accordingly, this response state may be determined beforetreatment and at various points during treatment of the individual withthe agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,polynucleotide, small molecule, or other drug candidate identified bythe screening assays described herein) including the steps of (i)obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression of aCVHG protein or mRNA in the pre-administration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the CVHG protein or mRNA in thepost-administration samples; (v) comparing the level of expression oractivity of the CVHG protein or mRNA in the pre-administration samplewith the CVHG protein or mRNA the post administration sample or samples;and (vi) altering the administration of the agent to the subjectaccordingly. According to such an embodiment, CVHG expression oractivity may be used as an indicator of the effectiveness of an agent,even in the absence of an observable phenotypic response.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk for, susceptible to or diagnosedwith CVH. With regard to both prophylactic and therapeutic methods oftreatment, such treatments may be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, includes the application of genomicstechnologies such as gene sequencing, statistical genetics, and geneexpression analysis to drugs in clinical development and on the market.More specifically, the term refers the study of how a subject's genesdetermine his or her response to a drug (e.g., a subject's “drugresponse phenotype” or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the CVHPP molecules ofthe present invention or CVHPP modulators (e.g., agonists orantagonists) according to that individual's drug response.Pharmacogenomics allows a clinician or physician to target prophylacticor therapeutic treatments to subjects who will most benefit from thetreatment and to avoid treatment of subjects who will experience toxicdrug-related side effects.

Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject CVH associated with aberrant CVHG expression or activity, byadministering to the subject anCVHG product or an agent which modulatesCVHG protein expression or activity.

Subjects at risk for CVH which is caused or contributed to by aberrantCVHG expression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.

Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the differential CVHGprotein expression, such that CVH is prevented or, alternatively,delayed in its progression. Depending on the type of CVHG aberrancy(e.g., typically a modulation outside the normal standard deviation),for example, a CVHG product, CVHG agonist or antagonist agent can beused for treating the subject. The appropriate agent can be determinedbased on screening assays described herein.

Therapeutic Methods

Another aspect of the invention pertains to methods of modulating CVHGprotein expression or activity for therapeutic purposes. Accordingly, inan exemplary embodiment, the modulatory method of the invention involvescontacting a cell with an agent that modulates one or more of theactivities of a CVHG product activity associated with the cell. An agentthat modulates CVHG product activity can be an agent as describedherein, such as a polynucleotide (e.g., an antisense molecule) or apolypeptide (e.g., a dominant-negative mutant of a CVHPP), anaturally-occurring target molecule of a CVHPP (e.g., a CVHPPsubstrate), an anti-CVHPP antibody, a CVHG modulator (e.g., agonist orantagonist), a peptidomimetic of a CVHG protein agonist or antagonist,or other small molecules.

The invention further provides methods of modulating a level ofexpression of a CVHG of the invention, comprising administration to asubject having CVH, a variety of compositions which correspond to theCVHGs of Tables 3-8, including proteins or antisense oligonucleotides.The protein may be provided by further providing a vector comprising apolynucleotide encoding the protein to the cells. Alternatively, theexpression levels of the CVHGs of the invention may be modulated byproviding an antibody, a plurality of antibodies or an antibodyconjugated to a therapeutic moiety. Treatment with the antibody mayfurther be localized to the tissue comprising CVH. In another aspect,the invention provides methods for localizing a therapeutic moiety toCVH tissue or cells comprising exposing the tissue or cells to anantibody which is specific to a protein encoded by the CVHGs of theinvention. This method may therefore provide a means to inhibitexpression of a specific gene corresponding to a CVHG listed in Tables3-8.

Determining Efficacy of a Test Compound or Therapy

The invention also provides methods of assessing the efficacy of a testcompound or therapy for inhibiting CVH in a subject. These methodsinvolve isolating samples from a subject suffering from CVH, who isundergoing treatment or therapy, and detecting the presence, quantity,and/or activity of one or more CVHGs of the invention in the firstsample relative to a second sample. Where the efficacy of a testcompound is determined, the first and second samples are preferablysub-portions of a single sample taken from the subject, wherein thefirst portion is exposed to the test compound and the second portion isnot. In one aspect of this embodiment, the CVHG is expressed at asubstantially decreased level in the first sample, relative to thesecond. Most preferably, the level of expression in the first sampleapproximates (i.e., is less than the standard deviation for normalsamples) the level of expression in a third control sample, taken from acontrol sample of normal tissue. This result suggests that the testcompound inhibits the expression of the CVHG in the sample. In anotheraspect of this embodiment, the CVHG is expressed at a substantiallyincreased level in the first sample, relative to the second. Mostpreferably, the level of expression in the first sample approximates(i.e., is less than the standard deviation for normal samples) the levelof expression in a third control sample, taken from a control sample ofnormal tissue. This result suggests that the test compound augments theexpression of the CVHG in the sample.

Where the efficacy of a therapy is being assessed, the first sampleobtained from the subject is preferably obtained prior to provision ofat least a portion of the therapy, whereas the second sample is obtainedfollowing provision of the portion of the therapy. The levels of CVHGproduct in the samples are compared, preferably against a third controlsample as well, and correlated with the presence, or risk of presence,of CVH. Most preferably, the level of CVHG product in the second sampleapproximates the level of expression of a third control sample. In thepresent invention, a substantially decreased level of expression of aCVHG indicates that the therapy is efficacious for treating CVH.

Pharmacogenomics

The CVHG protein and polynucleotide molecules of the present invention,as well as agents, inhibitors or modulators which have a stimulatory orinhibitory effect on CVHG expression or activity as identified by ascreening assay described herein, can be administered to individuals totreat (prophylactically or therapeutically) CVH associated with aberrantCVHG activity.

In conjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a CVHG product(polynucleotide or polypeptide) or CVHG modulator as well as tailoringthe dosage and/or therapeutic regimen of treatment with a CVHG productor CVHG modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. In general, two types of pharmacogeneticconditions can be differentiated. Genetic conditions transmitted as asingle factor altering the way drugs act on the body (altered drugaction) or genetic conditions transmitted as single factors altering theway the body acts on drugs (altered drug metabolism). Thesepharmacogenetic conditions can occur either as rare genetic defects oras naturally-occurring polymorphisms. For example, glucose-6-phosphatedehydrogenase deficiency (G6PD) is a common inherited enzymopathy inwhich the main clinical complication is hemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association,” relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related sites (e.g., a “bi-allelic” gene marker map which consistsof 60,000-100,000 polymorphic or variable sites on the human genome,each of which has two variants). Such a high-resolution genetic map canbe compared to a map of the genome of each of a statisticallysubstantial number of subjects taking part in a Phase II/III drug trialto identify genes associated with a particular observed drug response orside effect. Alternatively, such a high resolution map can be generatedfrom a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, an “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, an SNP may occur once per every 1,000 bases of DNA.An SNP may be involved in a disease process. However, the vast majorityof SNPs may not be disease associated. Given a genetic map based on theoccurrence of such SNPs, individuals can be grouped into geneticcategories depending on a particular pattern of SNPs in their individualgenome. In such a manner, treatment regimens can be tailored to groupsof genetically similar individuals, taking into account traits that maybe common among such genetically similar individuals. Thus, mapping ofthe CVHGs of the invention to SNP maps of CVH patients may allow easieridentification of these genes according to the genetic methods describedherein.

Alternatively, a method termed the “candidate gene approach,” can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug target is known (e.g., a CVHG ofthe present invention), all common variants of that gene can be fairlyeasily identified in the population and it can be determined if havingone version of the gene versus another is associated with a particulardrug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYPZC19) has provided an explanation as to why some subjectsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer and poor metabolizer. Theprevalence of poor metabolizer phenotypes is different among differentpopulations. For example, the gene coding for CYP2D6 is highlypolymorphic and several mutations have been identified in poormetabolizers, which all lead to the absence of functional CYP2D6. Poormetabolizers of CYP2D6 and CYP2C19 quite frequently experienceexaggerated drug response and side effects when they receive standarddoses. If a metabolite is the active therapeutic moiety, poormetabolizers show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., CVHG expression inresponse to a CVHG modulator of the present invention) can give anindication whether gene pathways related to toxicity have been turnedon.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a CVHG product orCVHG modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

Pharmaceutical Compositions

The invention is further directed to pharmaceutical compositionscomprising the test compound, or bioactive agent, or an CVHG modulator(i.e., agonist or antagonist), which may further include a CVHG product,and can be formulated as described herein. Alternatively, thesecompositions may include an antibody which specifically binds to a CVHGprotein of the invention and/or an antisense polynucleotide moleculewhich is complementary to a CVHG polynucleotide of the invention and canbe formulated as described herein.

One or more of the CVHGs of the invention, fragments of CVHGs, CVHGproducts, fragments of CVHG products, CVHG modulators, or anti-CVHPPantibodies of the invention can be incorporated into pharmaceuticalcompositions suitable for administration.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, solubilizers, fillers,stabilizers, binders, absorbents, bases, buffering agents, lubricants,controlled release vehicles, diluents, emulsifying agents, humectants,lubricants, dispersion media, coatings, antibacterial or antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well-known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary agents can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide orpolynucleotide corresponding to a CVHG of the invention. Such methodscomprise formulating a pharmaceutically acceptable carrier with an agentwhich modulates expression or activity of a CVHG. Such compositions canfurther include additional active agents. Thus, the invention furtherincludes methods for preparing a pharmaceutical composition byformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a CVHG and one or more additionalbioactive agents.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),intraperitoneal, transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine; propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfate; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, theinjectable composition should be sterile and should be fluid to theextent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requitedparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a fragment of a CVHPP or an anti-CVHPP antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the bioactive compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the therapeutic moieties, which may contain abioactive compound, are prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from e.g. Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form, as used herein, includesphysically discrete units suited as unitary dosages for the subject tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that includes the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The CVHGs of the invention can be inserted into gene delivery vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous administration, intraportaladministration, intrabiliary administration, intra-arterialadministration, direct injection into the liver parenchyma, byintramuscular injection, by inhalation, by perfusion, or by stereotacticinjection. The pharmaceutical preparation of the gene therapy vector caninclude the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Kits

The invention also encompasses kits for detecting the presence of a CVHGproduct in a biological sample, the kit comprising reagents forassessing expression of the CVHGs of the invention. Preferably, thereagents may be an antibody or fragment thereof, wherein the antibody orfragment thereof specifically binds with a protein corresponding to aCVHG from Table 4. For example, antibodies of interest may be preparedby methods known in the art. Optionally, the kits may comprise apolynucleotide probe wherein the probe specifically binds with atranscribed polynucleotide corresponding to a CVHG selected from thegroup consisting of the CVHGs listed in Tables 3-8. The kits may alsoinclude an array of CVHGs arranged on a biochip, such as, for example, aGeneChip®. The kit may contain means for determining the amount of theCVHG protein or mRNA in the sample; and means for comparing the amountof the CVHG protein or mRNA in the sample with a control or standard.The compound or agent can be packaged in a suitable container. The kitcan further comprise instructions for using the kit to detect CVHGprotein or polynucleotide

The invention further provides kits for assessing the suitability ofeach of a plurality of compounds for inhibiting CVH in a subject. Suchkits include a plurality of compounds to be tested, and a reagent (i.e.,antibody specific to corresponding proteins, or a probe or primerspecific to corresponding polynucleotides) for assessing expression of aCVHG listed in Tables 3-8.

Arrays and Biochips

The invention also includes an array comprising a panel of CVHGs of thepresent invention. The array can be used to assay expression of one ormore genes in the array.

It will be appreciated by one skilled in the art that the panels ofCVHGs of the invention may conveniently be provided on solid supports,such as a biochip. For example, polynucleotides may be coupled to anarray (e.g., a biochip using GeneChip® for hybridization analysis), to aresin (e.g., a resin which can be packed into a column for columnchromatography), or a matrix (e.g., a nitrocellulose matrix for Northernblot analysis). The immobilization of molecules complementary to theCVHG(s), either covalently or noncovalently, permits a discrete analysisof the presence or activity of each CVHG in a sample. In an array, forexample, polynucleotides complementary to each member of a panel ofCVHGs may individually be attached to different, known locations on thearray. The array may be hybridized with, for example, polynucleotidesextracted from a blood or colon sample from a subject. The hybridizationof polynucleotides from the sample with the array at any location on thearray can be detected, and thus the presence or quantity of the CVHG andCVHG transcripts in the sample can be ascertained. In a preferredembodiment, an array based on a biochip is employed. Similarly, Westernanalyses may be performed on immobilized antibodies specific for CVHPPshybridized to a protein sample from a subject.

It will also be apparent to one skilled in the art that the entire CVHGproduct (protein or polynucleotide) molecule need not be conjugated tothe biochip support; a portion of the CVHG product or sufficient lengthfor detection purposes (i.e., for hybridization), for example a portionof the CVHG product which is 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 100 or more nucleotides or amino acids in length may besufficient for detection purposes.

In one embodiment, the array can be used to assay gene expression in atissue to ascertain tissue specificity of genes in the array. In thismanner, a large number of genes can be simultaneously assayed forexpression. This allows an expression profile to be developed showing abattery of genes specifically expressed in one or more tissues at agiven point in time. In one embodiment the invention provides a kitcomprising a brochure which comprises at least 5, more preferably 10,more preferably or more CVHGs, and the same CVHGs in computer readableform.

In addition to such qualitative determination, the invention allows thequantitation of gene expression in the biochip. Thus, not only tissuespecificity, but also the level of expression of a battery of CVHGs inthe tissue is ascertainable. Thus, CVHGs can be grouped on the basis oftheir tissue expression per se and level of expression in that tissue.As used herein, a “normal level of expression” refers to the level ofexpression of a gene provided in a control sample, typically the controlis taken from either a non-diseased animal or from a subject who has notsuffered from CVH. The determination of normal levels of expression isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue or cell type can be perturbedand the effect on gene expression in a second tissue or cell type can bedetermined. In this context, the effect of one cell type on another celltype in response to a biological stimulus can be determined. Such adetermination is useful, for example, to know the effect of cell-cellinteraction at the level of gene expression. If an agent is administeredtherapeutically to treat one cell type but has an undesirable effect onanother cell type, the invention provides an assay to determine themolecular basis of the undesirable effect and thus provides theopportunity to co-administer a counteracting agent or otherwise treatthe undesired effect. Similarly, even within a single cell type,undesirable biological effects can be determined at the molecular level.Thus, the effects of an agent on expression of other than the targetgene can be ascertained and counteracted.

In another embodiment, the arrays can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment and differentiation, disease progression, in vitroprocesses, such as cellular transformation and activation.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

Importantly, the invention provides arrays useful for ascertainingdifferential expression patterns of one or more genes identified indiseased tissue versus non-diseased tissue. This provides a battery ofgenes that serve as a molecular target for diagnosis or therapeuticintervention. In particular, biochips can be made comprising arrays notonly of the CVHGs listed in Tables 3-8, but of CVHGs specific tosubjects suffering from specific manifestations or stages of thedisease.

In general, the probes are attached to the biochip in a wide variety ofways, as will be appreciated by those in the art. As described herein,the nucleic acids can either be synthesized first, with subsequentattachment to the biochip, or can be directly synthesized on thebiochip.

The biochip comprises a suitable solid substrate. By “substrate” or“solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of the nucleic acid probesand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates are very large,and include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, etc.

Generally the substrate is planar, although as will be appreciated bythose in the art, other configurations of substrates may be used aswell. For example, the probes may be placed on the inside surface of atube, for flow-through sample analysis to minimize sample volume.Similarly, the substrate may be flexible, such as a flexible foam,including closed cell foams made of particular plastics.

In a preferred embodiment, the surface of the biochip and the probe maybe derivatized with chemical functional groups for subsequent attachmentof the two. Thus, for example, the biochip is derivatized with achemical functional group including, but not limited to, amino groups,carboxy groups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the probes can beattached using functional groups on the probes. For example, nucleicacids containing amino groups can be attached to surfaces comprisingamino groups. Linkers, such as homo- or hetero-bifunctional linkers, mayalso be used.

In an embodiment, the oligonucleotides are synthesized as is known inthe art, and then attached to the surface of the solid support. As willbe appreciated by those skilled in the art, either the 5′ or 3′ terminusmay be attached to the solid support, or attachment may be via aninternal nucleoside.

In an additional embodiment, the immobilization to the solid support maybe very strong, yet non-covalent. For example, biotinylatedoligonucleotides can be made, which bind to surfaces covalently coatedwith streptavidin, resulting in attachment.

Alternatively, the oligonucleotides may be synthesized on the surface,as is known in the art. For example, photoactivation techniquesutilizing photopolymerization compounds and techniques are used. In apreferred embodiment, the nucleic acids can be synthesized in situ,using well known photolithographic techniques.

Modifications to the above-described compositions and methods of theinvention, according to standard techniques, will be readily apparent toone skilled in the art and are meant to be encompassed by the invention.This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and Tables are incorporated hereinby reference.

EXAMPLES Example 1 Methods (a) Visceral Sensitization Model

New born rats were sensitized by infusion of 0.2 mls of 0.5% acetic acidinto the colon at P10, control animals received saline.

(b) CNI1493 Treatment:

At eight weeks of age, rats in four treatment groups: control+vehicle(n=3), control+cni-1493 (n=3), sensitized+vehicle (n=8),sensitized+cni-1493 (n=8) were tested after administration of drug(dissolved in 2.5% mannitol, injected i.p. at 5 mg/kg for four days) forsensitivity to colorectal distention (CRD). Vehicle injected animalsreceived 2.5% mannitol in water.

(c) CRD Protocol for Measuring Colonic Sensitivity in Adult Rats

For electromyographic (EMG) measurements of visceromotor responses,under anesthesia (Nembutal, 50 mg/kg i.p.), two electrodes wereimplanted in the abdominal wall muscles and externalized behind thehead. Rats were allowed a one week recovery time from this surgery.Under mild sedation (Brevital, 5 ml of 1% ip), a 7 cm flexible balloonconstructed from a surgical glove finger attached to tygon tubing wasinserted into the descending colon and rectum via the anus and held inplace by taping the tubing to the tail. Rats were housed in small Lucitecubicles (20×8×8 cm) and were allowed to adapt for one hour. CRD wasperformed by rapidly inflating the balloon to constant pressure.Pressure was measured using a sphygmomanometer connected to a pressuretransducer. Rats were given graded CRD (20, 40, 60, 80 mm Hg) appliedfor 20 seconds every 4 minutes.

The behavioral measurements consisted of visual observation of theabdominal withdrawal reflex (AWR) by blinded observers and theassignment of an AWR score:

0, no response;

1, brief head movement followed by immobility;

2, contraction of abdominal muscles;

3, lifting of abdomen;

4, body arching and lifting of pelvic structures.

(d) Evaluation of the Colon for Inflammation/Damage

Descending colons were placed in 10% buffered formalin and portions werefrozen for myeloperoxidase (MPO) assays. Haematoxilin & Eosin stainedparaffin sections will be scored for inflammation by a pathologist.

(e) Tissue Isolation and RNA Preparation

Colons and S1 dorsal root ganglia were snap frozen in liquid nitrogen.Colon tissue RNA and S1 dorsal root ganglia RNA was prepared usingRNAzol.

(f) Target Labeling and Gene Chip Array Analysis

An in vitro transcription reaction was performed using 500 ng cDNA whichcontained a T7 RNA polymerase promoter incorporated during RNAamplification. The cRNA or Target RNAs produced during this in vitrotranscription reaction were labeled with biotin. Biotin-labeled TargetRNAs were fragmented to a mean size of 200 bases to facilitate theirhybridization to probe sequences on the Gene Chip (Affymetrix) array.Each Target RNA sample was initially hybridized to a test array. Thisarray contains a set of probes representing genes that are commonlyexpressed in the majority of cells (example Rat: actin, GAPDH,hexokinase, 5S rRNA, and B1/B2 repetitive elements). Test arraysconfirmed the successful labeling of the target RNAs and prevented theuse of degraded or non-representative target RNA samples. Success andconsistency of RNA amplification between samples were also confirmedwith these test arrays.

Hybridization of Gene Chip (Affymetrix) arrays was performed at 45° C.for 16 hours in 0.1 M MES pH6.6, 1 M sodium chloride, 0.02 M EDTA and0.01% Tween 20. Four prokaryotic genes (bio B, bio C and bio D from theE. coli biotin synthesis pathway and the cre recombinase gene from P1bacteriophage) were added to the hybridization cocktail as internalcontrols. These control RNAs were used to normalize expression levelsbetween experiments. Because the control RNAs were added at varying copynumber (Bio B, 1.5 pM; Bio C, 5 pM; Bio D, 25 pM and cre, 100 pM) theywere also used in estimating relative abundance of RNA transcripts inthe sample. Arrays were washed using both non-stringent (1 M NaCl, 25°C.) and stringent (1 M NaCl, 50° C.) conditions prior to staining withphycoerythrin streptavidin (10 ug/ml final). Gene Chip arrays werescanned using a Gene Array Scanner (Hewlett Packard) and analyzed usingthe Affymetric Gene Chip Analysis Suite 5.0 software.

Gene expression profiles were produced from Affymetrix rat genome 230Achips.

Single array analysis for each chip was performed by AffymetrixMicroarray Suite (MAS) software to produce a detection call, present,absent or marginal and a signal intensity value for each gene that is arelative measure of abundance of the transcript. For comparison ofsignal intensity values between chips, all chips were scaled to anaverage intensity of 500. Genes called “Absent” across all chips andgenes without a |Fold change|≧2.0 in at least one of the pairwisecomparisons of chips from different treatment groups were excluded. Theprobe sets with absolute call “Absent” across all chips and |Foldchange|<2.0 in all of the possible pairwise comparisons were filteredout. These filters were applied as the first level filters to reduce thenoise from the dataset. ANOVA was performed on the filtered dataset.Significant changes in gene expression were detected by analyzing signalintensity values by two-way ANOVA with 99% confidence limits. Genes weresubjected to cluster analysis to identify genes associated withsensitization and treatment.

(g) Quantitative RT-PCR

Primer or primer/probe sets were designed using Primer Express software(Applied Biosystems, Foster City, Calif.) such that the amplimer spannedan intron/exon boundary where possible. Where ESTs were homologous toknown genes, the sequence of the known gene was used. RT-PCR wasperformed using Applied Biosystems reagents and kits: Taqman ReverseTranscription Reagents N8080234 and Taqman PCR Core Reagents N8080228.PCR was performed on a GeneAmp 5700 Sequence Detection System (AppliedBiosystems). Machine default PCR program was used: 2 min at 50° C., 10min at 95° C., 45 cycles: 95° C. 15 sec, 60° C. 1 min. Fold change wascalculated using delta Ct and/or relative standard curve proceduresusing GAPDH or b-actin as normalizers for colon genes and PGP9.5 forDRG. Data is expressed as fold change relative to control, vehicletreated values.

Primer Probe Sequences (RAT):

Desmin NM_022531 Forward primer (FP) GTGGAGCGTGACAACCTGATAGReverse primer (RP) TGCGCTCTAGGTCAATTCGACEBP/delta NM_013154 Mar. 3, 2004 SYBR green FP CCGCCCGAATTGCTACAGT RPAGTCTGTCGGAAAAGTCTTTTCTACAA EST homologous to F-box proteins FPCCGTGTGCAAGTGTGTAGCAT RP GCCGCAGCCCGAAAG PEP19 FP GCTGGAGCAACCAATGGAAARP TCTGTCTCTGGTGCATCCATGT Probe TCT TCT TGG ACC TTC TT CTG CCC ATC ATT

Insulin-Like Growth Factor Binding Protein 2 Colon 71 NM_(—)013122

FP 1001-1022 CAACCTCAAACAGTGCAAGATG RP 1182-1163 TGGTTTACTGCACCCTTTGGMetalloproteinase ADAMTS-1 FP AGGGACCGGAAGTTACTTCCA RPCAGGTGTGGGAGCCACATAA Tubulin, beta 3 FP GGGCCTTTGGACACCTATTCA RPGCCCTTTGGCCCAGTTGT PROBE C ACC ACT CTG ACC GAA GAT AAA GTT GTC AGGArgalb NM_053770 Feb. 19, 2004 FP GAATCCCCACAGCCATTAGAAC RPGCGAGTTGTACAGACCTGCATT Probe ACA TGT CTG TGT CCT CAT CCG GCT TGTStathmin-like 2 (scgn10) FP TCGGAAGCTCCACGAACTCT RP CTCGCTCGTGCTCCCTCTT

(f) MPO Assay

Myeloperoxidase (MPO) assays were performed as described (Bhatia et al.,Proc Natl Acad Sci USA 95:4760-4756, 1998). Frozen colon was homogenizedin 20 mM phosphate buffer pH 7.4 and centrifuged at 10,000×g for 10minutes at 4° C. Pellet was resuspended in 50 mM phosphate buffer, pH6.0 containing 0.5% hexadecyltrimethylammonium bromide. Samples weresubjected to one cycle of freeze thawing, were incubated at 60° C. for 2hrs and centrifuged at 10,000×g for 5 min at 4° C. Change in absorbanceat 655 nM was measured in reaction mix containing 1.6 mM TMB (Sigma) and0.3 mM H₂O₂ on a Beckman DU-64 spectrophotometer. Activity wasnormalized to protein as measured in the extracts. Protein was measuredby the BCA method (Pierce, Rockford, Ill.). Activity was expressed asthe change in absorbance/min/mg protein.

Example 2 Chemical Treatment of Colon of P10 Rats Produces CVH

Previous studies showed that either mechanical irritation or treatmentwith mustard oil of the colons of young rats between P7 and P12 producedchronic visceral hyperalgesia (ref). To determine whether treatment ofthe colon of young rats with acetic acid would produce visceralhyperalgesia in adults, the colon of P10 rats was infused with 0.5%acetic acid; control littermates received saline. Rats were tested at 8weeks of age for sensitivity to CRD and colons were examined forhistopathological evidence of inflammation. Adult rats treated withacetic acid on P10 exhibited increased sensitivity to CRD compared tocontrols (FIG. 1). Data was analyzed by two-way repeated measures ANOVAwith distention pressure as the repeated factor and P10 treatment as abetween group factor. There was a significant effect of P10 treatment (F1, 12.98) p<0.003, of distention pressure (F 7, 89.9) p<0.001, and therewas a significant interaction between distention pressure and P10treatment (F 7, 4.04) p<0.001. Means were compared with a Tukey test.Significant differences between AA treated and controls were found atdistention pressures of 30 (p=0.004), 40 (p<0.001), 50 (p<0.001), 60(p=0.001) and 70 (p=0.035) mm Hg. Chemical treatment of colon of P10rats produces chronic visceral hypersensitivity.

Example 3 Effect of CNI1493 on Visceral Hypersensitivity

Rats were treated for 4 days with CNI1493 and sensitivity to CRD wasmeasured after treatment. Sensitivity to CRD was significantly reducedby drug treatment F(1, 5.46) p=0.05 (FIG. 2) but was unchanged invehicle treated rats. Data was analyzed by two-way repeated measuresANOVA with distention pressure as the repeated factor and drug treatmentas a between group factor. There was a significant effect of CNI1493treatment (F 1, 16.96) p=0.001, of distention pressure (F 7, 28.55)p<0.001, but there was no significant interaction between distentionpressure and CNI-1493 treatment (F 7, 1.94) p=0.071. Means were comparedwith a Tukey test. Significant differences between CNI-1493 treated andcontrols were found at distention pressures of 20 (P=0.001), 30(p=0.003), 40 (p<0.001), 50 (p=0.001), 60 (p<0.001) and 70 (p=0.015) and80 (p=0.007) mm Hg.

To determine whether P10 colon irritation produced chronic inflammation,H&E stained colon sections were examined for signs of inflammation andMPO activity in colon extracts was examined. No differences inappearance of the sections were noted between treatment groups and noovert signs of inflammation were observed. Mucosal architecture wasnormal, there was no cellular infiltrate, and there was no depletion ofgoblet cells. No differences were observed in colon histopathology (FIG.3A-3D). There was a significant increase in MPO activity in thesensitized rats compared to controls (FIG. 3E). CNI-1493 treatmentsignificantly lowered MPO activity in sensitized rats. These findingssuggest that there was a low grade inflammation or increased lymphocytespresent in sensitized rats.

Example 4 Gene Expression Profiles in Colon and S1 DRG

To determine the long term effects of P10 colon irritation and ofsubsequent CNI1493 treatment on gene expression in the colon, geneexpression profiles of rat colons from each treatment group derived fromAffymetrix rat genome 230A chips were subjected to cluster analysis toidentify genes associated with sensitization and treatment. The analysisrevealed that 114 genes were differentially expressed in sensitized/CVHcolon (Table 3), 76 genes were differentially expressed insensitized/CVH S1 DRG (Table 4), 660 genes were differentially expressedin CNI1493-treated colon (Table 5), and 137 genes were differentiallyexpressed in CNI1493-treated S1 DRG (Table 6). Since CNI1493 treatmentameliorates CVH in the sensitized animals, genes differentiallyregulated by CNI1493 may also be related to the etiology of CVH.Accordingly, genes listed in Tables 3-8 are designated as CVH-relatedgenes (CVHGs). Expression levels of a subset of genes from each groupwere confirmed by quantitative RT-PCR (Table 8).

TABLE 8 Comparison of chip results with RT-PCR Gene sen + sen + sen +veh sen + CNI veh RT- CNI RT- Chip PCR Chip PCR desmin 2.6 1.7 1.3 0.8CCAAT/enhancerbinding 2.4 2.2 2.3 1.6 (C/EBP) delta EST similar to F-boxproteins 2.4 1.9 1.2 0.6 neuron specific protein PEP-19 2.3 2.1 1.1 1.1Insulin-like growth factor binding 2.2 2.6 0.9 0.7 protein 2 ADAMTS1 2.12.0 1.1 0.8 tubulin, beta 3 2.1 2.9 1.5 1.3 ArgBP2 1.7 2.9 0.8 1.8scgn10 1.5 1.3 1.1 0.4 BDNF 1.7 1.9 1.7 2.1 phosphodiesterase 3B 1.8 1.61.1 1.2 Trek2 2.1 1.5 1.7 0.9 TrkA precursor 0.7 0.8 0.7 0.7 interleukin1 receptor, type 1 0.5 1.1 0.6 0.8 elongation factor 2 kinase 0.4 1.60.8 1.5

1. A method for detecting CVH in a subject, said method comprising: (a)contacting a biological sample with an agent that specifically binds toa polypeptide encoded by a gene listed in Tables 3-8 or a homologthereof; (b) determining a level of binding of the agent to thepolypeptide; (c) comparing the level of binding of the agent in thebiological sample to a level of binding of the agent in a normal controlsample; and (d) producing a diagnosis based on a result from step (c).2. The method of claim 1, wherein said polypeptide comprises an aminoacid sequence recited in any one of SEQ ID NOS:34-66.
 3. The method ofclaim 1, wherein the agent is an antibody directed against thepolypeptide.
 4. A method for detecting CVH in a subject, said methodcomprising the steps of: (a) determining a level of a transcribedpolynucleotide in a biological sample obtained from the subject, whereinthe transcribed polynucleotide is transcribed from a gene listed inTables 3-8 or homolog thereof; (b) comparing the level of thetranscribed polynucleotide in the biological sample to a normal level ofthe transcribed polynucleotide; and (c) producing a diagnosis based on aresult from step (b).
 5. The method of claim 4, wherein said transcribedpolynucleotide comprises a nucleic acid sequence recited in any one ofSEQ ID NOS:1-33, or a complement of any of the foregoing nucleic acidsequences.
 6. The method of claim 4, wherein the transcribedpolynucleotide is an mRNA.
 7. A method for detecting CVH in a subject,said method comprising the steps of: (a) determining an expressionpattern of polypeptides in a biological sample, said polypeptides areencoded by the genes listed in Tables 3-8 or homologs thereof; (b)comparing the expression pattern of polypeptides obtained in (a) to anormal expression pattern of polypeptides; and (c) producing a diagnosisbased on a result from step (b).
 8. The method of claim 7, wherein thepolypeptides comprise polypeptide sequences recited in SEQ ID NOS:34-66.9. The method of claim 7, wherein the expression pattern of polypeptidesin the biological sample is determined using antibodies directed againstthe polypeptides.
 10. The method of claim 7, wherein the expressionpattern of polypeptides is determined by measuring expression levels ofat least three polypeptides.
 11. A method for detecting CVH in asubject, said method comprising the steps of: (a) determining anexpression pattern of mRNAs in a biological sample, said mRNAs aretranscribed from the genes listed in Tables 3-8 or homologs thereof; (b)comparing the expression pattern of mRNAs obtained in (a) to a normalexpression pattern of mRNAs; and (c) producing a diagnosis based on aresult from step (b).
 12. The method of claim 11, wherein said mRNAscomprise polynucleotide sequences recited in SEQ ID NOS:1-33.
 13. Themethod of claim 11, wherein the expression pattern of mRNAs isdetermined by measuring expression levels of at least three mRNAs.14.-27. (canceled)
 24. A biochip comprising at least one of: (a) apolynucleotide comprising a sequence that hybridizes to a gene listed inTables 3-8 or a homolog thereof; (b) a polypeptide comprising at least aportion of a sequence encoded by a gene listed in Tables 3-8.
 25. Thebiochip of claim 24, wherein said polynucleotide comprises a sequencethat hybridizes to any one of SEQ ID NOS: 1-33, and wherein saidpolypeptide comprising any one of SEQ ID NOS:34-66.
 26. A diagnostic kitfor CVH, said kit comprising at least one of: a polynucleotide probe,wherein the probe specifically binds to a polynucleotide transcribedfrom a gene listed in Tables 3-8 or a homolog thereof; and an antibodycapable of immunospecific binding to a polypeptide encoded by a genelisted in tables 3-8.
 27. The diagnostic kit of claim 26, wherein theprobe specifically binds to a transcribed polynucleotide comprising anyone of SEQ ID NOS:1-33.
 28. The diagnostic kit of claim 26, wherein theantibody specifically binds to a polypeptide comprising any one of SEQID NOS:34-66. 29.-31. (canceled)